Nkp30 receptor targeted therapeutics

ABSTRACT

The invention is directed to T cells and other cells that express chimeric NK-p30 receptors (“chimeric NKp30 T cells”), methods of making and using chimeric NKp30 T cells, and methods of using these chimeric NKp30 T cells, isolated populations thereof, and compositions comprising the same. In another aspect, said chimeric NKp30 T cells are further designed to express a functional non-TCR receptor. The disclosure also pertains to methods of making said chimeric NKp30 T cells, and methods of reducing or ameliorating, or preventing or treating, diseases and disorders using said chimeric NKp30 T cells, populations thereof, or compositions comprising the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Utility application Ser. No.15/830,605 filed Dec. 4, 2017, now U.S. Pat. No. 10,682,378, which is adivisional of U.S. application Ser. No. 14/342,060, filed Nov. 24, 2014,now U.S. Pat. No. 9,833,476, which is a 35 U.S.C. 371 United StatesNational Phase Application of PCT Application PCT/US2012/053511, filedAug. 31, 2012 and published as WO 2013/033626 on Mar. 7, 2013, whichclaims priority to U.S. Provisional Application 61/529,410 filed Aug.31, 2011, each of which is hereby incorporated by reference in itsentirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under contract number CAawarded by the National Institutes of Health. The government has certainrights in the invention.

SEQUENCE LISTING

The sequence listing in the filed named “1148307o001203.txt” having asize of 84,226 bytes that was created May 26, 2020, is herebyincorporated by reference in its entirety.

BACKGROUND Field

The present disclosure is directed to T cells that express chimericNKp30 receptors (“chimeric NKp30 T cells”), methods of making and usingchimeric NKp30 T cells, and methods of using these chimeric NKp30 Tcells to address diseases and disorders. In one aspect, the disclosurebroadly relates to chimeric NKp30 T cells, isolated populations thereof,and compositions comprising the same. In another aspect, said chimericNKp30 T cells are further designed to express a functional non-TCRreceptor. The disclosure also pertains to methods of making saidchimeric NKp30 T cells, and methods of reducing or ameliorating, orpreventing or treating, diseases and disorders, especially cancers,using said chimeric NKp30 T cells, populations thereof, or compositionscomprising the same.

Description of Related Art

The global burden of cancer doubled between 1975 and 2000, and cancer isexpected to become the leading cause of death worldwide by 2010.According to the American Cancer Society, it is projected to doubleagain by 2020 and to triple by 2030. Thus, there is a need for moreeffective therapies to treat various forms of cancer. Ideally, anycancer therapy should be effective (at killing cancerous cells),targeted (i.e. selective, to avoid killing healthy cells), permanent (toavoid relapse and metastasis), and affordable. Today's standards of carefor most cancers fall short in some or all of these criteria.

T cells, especially cytotoxic T cells, play important roles inanti-tumor immunity (Rossing and Brenner (2004) Mol. Ther. 10:5-18).Adoptive transfer of tumor-specific T cells into patients provides ameans to treat cancer (Sadelain, et al. (2003) Nat. Rev. Cancer3:35-45). However, the traditional approaches for obtaining largenumbers of tumor-specific T cells are time-consuming, laborious andsometimes difficult because the average frequency of antigen-specific Tcells in periphery is extremely low (Rosenberg (2001) Nature411:380-384; Ho, et al. (2003) Cancer Cell 3:431-437; Crowley, et al.(1990) Cancer Res. 50:492-498). In addition, isolation and expansion ofT cells that retain their antigen specificity and function can also be achallenging task (Sadelain, et al. (2003) supra). Genetic modificationof primary T cells with tumor-specific immunoreceptors, such asfull-length T cell receptors or chimeric T cell receptor molecules canbe used for redirecting T cells against tumor cells (Stevens, et al.(1995) J. Immunol. 154:762-771; Oelke, et al. (2003) Nat. Med.9:619-624; Stancovski, et al. (1993) J. Immunol. 151:6577-6582; Clay, etal. (1999) J. Immunol. 163:507-153). This strategy avoids the limitationof low frequency of antigen-specific T cells, allowing for facilitatedexpansion of tumor-specific T cells to therapeutic doses.

Natural killer (NK) cells are innate effector cells serving as a firstline of defense against certain viral infections and tumors (Biron, atal. (1999) Annu. Rev. Immunol. 17:189-220; Trinchieri (1989) Adv.Immunol. 47:187-376). They have also been implicated in the rejection ofallogeneic bone marrow transplants (Lanier (1995) Curr. Opin. Immunol.7:626-631; Yu, et al. (1992) Annu. Rev. Immunol. 10:189-214). Innateeffector cells recognize and eliminate their targets with fast kinetics,without prior sensitization. Therefore, NK cells need to sense if cellsare transformed, infected, or stressed to discriminate between abnormaland healthy tissues. According to the missing self phenomenon (Karre, etal. (1986) Nature (London) 319:675-678), NK cells accomplish this bylooking for and eliminating cells with aberrant major histocompatibilitycomplex (MHC) class I expression; a concept validated by showing that NKcells are responsible for the rejection of the MHC class I-deficientlymphoma cell line RMA-S, but not its parental MHC class I-positive lineRMA.

Natural killer (NK) cells can also attack tumor and virally infectedcells in the absence of MHC restriction, utilizing a combination ofsignals from activating and inhibitory receptors. One group ofactivating NK receptors are natural cytotoxicity receptors (NCRs), whichinclude NKp46 (NCR1), NKp44 (NCR2) and NKp30 (also called naturalcytotoxicity receptor 3 (NCR3) or CD337). These receptors areexclusively expressed on NK cells, which play important roles inNK-mediated tumor cell-killing.

NKp30 is an activating NK receptor that is involved in the NK-mediatedkilling of tumor cells. NKp30 recognizes ligands on tumor cells anddendritic cells. These ligands are highly expressed on a subset of tumorcells, but not most other normal cells. There is some evidence that somesubsets of dendritic cells may express these ligands in vitro. Inlaboratory mice, NKp30 is a pseudogene. NKp30 has been further describedin the literature including Brandt et al., J Exp Med. 2009 Jul. 6;206(7):1495-503; Byrd et al., PLoS One. 2007 Dec. 19; 2(12):e1339; andDelahaye et al., Nat Med. 2011 June; 17(6):700-7, each of which isincorporated by reference herein in its entirety.

Two cellular NKp30 receptor ligands have been identified: BAT3 andB7-H6. BAT3 is a nuclear protein, which is involved in the interactionwith P53 and induction of apoptosis after stress such as DNA damage.B7-H6 is a recently identified B7 family member. Structures of an NKp30ligand binding site and an NKp30-B7-H6 complex have been reported in theliterature (Li et al., J Exp Med. 2011 Apr. 11; 208(4):703-14; Joyce etal., Proc Natl Acad Sci USA. 2011 Apr. 12; 108(15):6223-8). Unlike BAT3,B7-H6 is expressed on the surface of tumor cells, but not normal cells.Thus, the NKp30 receptor-NKp30 ligand system provides a relativelyspecific system for immune cells to recognize tumor cells.

NKp30 associates with CD3ζ and FcRγ for signal transduction. A recentstudy shows that there exist three isoforms of NKp30 (i.e., A, B and C),which differs in signaling capacity in NK cells (Delahaye et al., NatMed. 2011 June; 17(6):700-7). Isoforms A and B were reported toefficiently interact with CD3ζ and are associated with good prognosis ofgastrointestinal stromal tumors, whereas isoform C poorly associate withCD3ζ and linked to poor prognosis. Specifically, isoform A wasdemonstrated to associate with CD3ζ upon NKp30 cross-linking, whereasisoform B was demonstrated to constitutively associate with CD3ζ.

Inhibitory receptors specific for MHC class I molecules have beenidentified in mice and humans. The human killer cell Ig-like receptors(KIR) recognize HLA-A, -B, or -C; the murine Ly49 receptors recognizeH-2K or H-2D; and the mouse and human CD94/NKG2 receptors are specificfor Qalb or HLA-E, respectively (Long (1999) Annu. Rev. Immunol.17:875-904; Lanier (1998) Annu. Rev. Immunol. 16:359-393; Vance, et al.(1998) J. Exp. Med. 188:1841-1848).

Activating NK cell receptors specific for classic MHC class I molecules,nonclassic MHC class I molecules or MHC class I-related molecules havebeen described (Bakker, et al. (2000) Hum. Immunol. 61:18-27). One suchreceptor is NKG2D (natural killer cell group 2D) which is a C-typelectin-like receptor expressed on NK cells, γδ-TcR+ T cells, and CD8+αβ-TcR+ T cells (Bauer, et al. (1999) Science 285:727-730). NKG2D isassociated with the transmembrane adapter protein DAP10 (Wu, et al.(1999) Science 285:730-732), whose cytoplasmic domain binds to the p85subunit of the PI-3 kinase.

In humans, two families of ligands for NKG2D have been described (Bahram(2000) Adv. Immunol. 76:1-60; Cerwenka and Lanier (2001) Immunol. Rev.181:158-169). NKG2D binds to the polymorphic MHC class I chain-relatedmolecules (MIC)-A and MICB (Bauer, et al. (1999) supra). These areexpressed on many human tumor cell lines, on several freshly isolatedtumor specimens, and at low levels on gut epithelium (Groh, et al.(1999) Proc. Nat. Acad. Sci. USA 96:6879-6884). NKG2D also binds toanother family of ligands designated the UL binding proteins (ULBP)-1,-2, -3, and -4 molecules (Cosman, et al. (2001) Immunity 14:123-133;Kubin, et al. (2001) Eur. J. Immunol. 31:1428-1437; Conejo-Garcia, J.R., F. Benencia, et al. (2003). “Letal, A tumor-associated NKG2Dimmunoreceptor ligand, induces activation and expansion of effectorimmune cells.” Cancer Biol Ther 2(4): 446-451). Although similar toclass I MHC molecules in their α1 and α2 domains, the genes encodingthese proteins are not localized within the MHC. Like MIC (Groh, et al.(1996) supra), the ULBP molecules do not associate with P2-microglobulinor bind peptides. The known murine NKG2D-binding repertoire encompassesthe retinoic acid early inducible-1 gene products (RAE-I) and therelated H60 minor histocompatibility antigen (Cerwenka, et al. (2000)Immunity 12:721-727; Diefenbach, et al. (2000) Nat. Immunol. 1:119-126).RAE-I and H60 were identified as ligands for mouse NKG2D by expressioncloning these cDNA from a mouse transformed lung cell line (Cerwenka, etal. (2000) supra). Transcripts of RAE-I are rare in adult tissues butabundant in the embryo and on many mouse tumor cell lines, indicatingthat these are oncofetal antigens.

Recombinant receptors containing an cytoplasmic domain for activating Tcells and an extracellular antigen-binding domain, which is typically asingle-chain fragment of a monoclonal antibody and is specific for atumor-specific antigen, have been reported for targeting tumors fordestruction. See, e.g., U.S. Pat. No. 6,410,319.

Baba et al. ((2000) Hum. Immunol. 61:1202-18) disclose KIR2DL1-CD3ζchain chimeric proteins. Further, WO 02/068615 (which describes priorwork by the present inventors) suggests fusion proteins of NKG2Dcontaining the external domain of NKG2D with a distinct DAP10interacting domain or with cytoplasmic domains derived from othersignaling molecules, for example CD28, for use in engineering cells thatrespond to NKG2D ligands and potentially create a system with enhancedsignaling capabilities.

Brandt et al., J Exp Med. 2009 Jul. 6; 206(7):1495-503 discloses use ofIL-2-producing DO11.10 mouse T cell hybridoma expressing a chimericreceptors (in which the intracytoplasmic domain of mouse CD3ζ was fusedeither to the extracellular portion of NKp30 or NKp46) as reporterconstructs in assays to evaluate recognition of ligands which weremeasured by detecting IL-2 secretion. However, the reference does notreport introduction of these constructs into normal T cells ortherapeutic use of these constructs (e.g., for treatment of cancer).

U.S. Pat. No. 5,359,046 discloses a chimeric DNA sequence encoding amembrane bound protein, wherein the chimeric DNA comprises a DNAsequence encoding a signal sequence which directs the membrane boundprotein to the surface membrane; a DNA sequence encoding a non-MHCrestricted extracellular binding domain of a surface membrane proteinselected from the group consisting of CD4, CD8, IgG and single-chainantibody that binds specifically to at least one ligand, wherein saidligand is a protein on the surface of a cell or a viral protein; atransmembrane domain from a protein selected from the group consistingof CD4, CD8, IgG, single-chain antibody, the CD3ζ chain, the CD3γ chain,the CD3δ chain and the CD3ε chain; and a cytoplasmic signal-transducingdomain of a protein that activates an intracellular messenger systemselected from the group consisting of the CD3ζ chain, the CD3γ chain,the CD3δ chain and the CD3ε chain, wherein the extracellular domain andcytoplasmic domain are not naturally joined together and the cytoplasmicdomain is not naturally joined to an extracellular ligand-bindingdomain, and when the chimeric DNA is expressed as a membrane boundprotein in a selected host cell under conditions suitable forexpression, the membrane bound protein initiates signaling in the hostcell.

Cellular immunotherapy has been shown to result in specific tumorelimination and has the potential to provide specific and effectivecancer therapy (Ho, W. Y. et al. 2003. Cancer Cell 3:1318-1328; Morris,E. C. et al. 2003. Clin. Exp. Immunol. 131:1-7; Rosenberg, S. A. 2001.Nature 411:380-384; Boon, T. and P. van der Bruggen. 1996. J. Exp. Med.183:725-729). T cells have often been the effector cells of choice forcancer immunotherapy due to their selective recognition and powerfuleffector mechanisms. T cells recognize specific peptides derived frominternal cellular proteins in the context of self-majorhistocompatibility complex (MHC) using their T cell receptors (TCR).

WO/2006/036445 (and its U.S. counterpart, now patented as U.S. Pat. No.7,924,298) discloses a chimeric receptor protein comprising a C-typelectin-like natural killer cell receptor, or a protein associatedtherewith, fused to an immune signaling receptor having animmunoreceptor tyrosine-based activation motif for reducing oreliminating a tumor. To the N-terminus of the C-type lectin-like NK cellreceptor is fused an immune signaling receptor having an immunoreceptortyrosine-based activation motif (TTAM),(Asp/Glu)-Xaa-Xaa-Tyr*-Xaa-Xaa(Ile/Leu)-Xaa₆₋₈-Tyr*-Xaa-Xaa-(Ile/Leu)which is involved in the activation of cellular responses via immunereceptors. Similarly, when employing a protein associated with a C-typelectin-like NK cell receptor, an immune signaling receptor can be fusedto the C-terminus of said protein. That publication additionallydiscloses that suitable immune signaling receptors for use in thechimeric receptor include, but are not limited to, the ζ chain of theT-cell receptor, the eta chain which differs from the ζ chain only inits most C-terminal exon as a result of alternative splicing of the ζmRNA, the δ, γ and ε chains of the T-cell receptor (CD3 chains) and theγ subunit of the FcR1 receptor. That publication further discloses thatthe immune signaling receptor may be CD3ζ (e.g., GENBANK accessionnumber NM_198053), or human Fcε receptor-γ chain (e.g., GENBANKaccession number M33195) or the cytoplasmic domain or a splicing variantthereof. Further exemplary chimeric receptors described in thatpublication include is a fusion between NKG2D and CD3ζ or DAP10 andCD3ζ.

It is recognized in the art that the TCR complex associates in precisefashion by the formation of dimers and association of these dimers(TCR-α/ρ, CD3-γ/ε, CD3-δ/ε, and CD3ζ dimer) into one TCR complex thatcan be exported to the cell surface. The inability of any of thesecomplexes to form properly can inhibit TCR assembly and expression(Call, M. E. et al., (2007) Nature Rev. Immunol., 7:841-850; Call, M. E.et al., (2005) Annu. Rev. Immunol., 23:101-125).

Particular amino acid residues in the respective TCR chains have beenidentified as important for proper dimer formation and TCR assembly. Inparticular, for TCR-α, these key amino acids in the transmembraneportion are arginine (for association with CD3ζ) and lysine (forassociation with the CD3-ε/δ dimer). For TCR-β, the key amino acid inthe transmembrane portion is a lysine (for association with CD3-ε/γdimer). For CD3-γ, the key amino acid in the transmembrane portion is aglutamic acid. For CD3-δ, the key amino acid in the transmembraneportion is an aspartic acid. For CD3-ε, the key amino acid in thetransmembrane portion is an aspartic acid. For CD3ζ, the key amino acidin the transmembrane portion is an aspartic acid (Call, M. E. et al.,(2007) Nature Rev. Immunol., 7:841-850; Call, M. E. et al., (2005) Annu.Rev. Immunol., 23:101-125).

Peptides derived from altered or mutated proteins in tumors can berecognized by specific TCRs. Several key studies have led to theidentification of antigens associated with specific tumors that havebeen able to induce effective cytotoxic T lymphocyte (CTL) responses inpatients (Ribas, A. et al. 2003. J. Clin. Oncol. 21:2415-2432). T celleffector mechanisms include the ability to kill tumor cells directly andthe production of cytokines that activate other host immune cells andchange the local tumor microenvironment. Theoretically, T cells couldidentify and destroy a tumor cell expressing a single mutated peptide.Adoptive immunotherapy with CTL clones specific for MARTI or gp100 withlow dose IL-2 has been effective in reduction or stabilization of tumorburden in some patients (Yee, C. et al. 2002. Proc. Natl. Acad. Sci. USA99:16168-16173). Other approaches use T cells with a defined anti-tumorreceptor. These approaches include genetically modifying CTLs with newantigen-specific T cell receptors that recognize tumor peptides and MHC,chimeric antigen receptors (CARs) derived from single chain antibodyfragments (scFv) coupled to an appropriate signaling element, or the useof chimeric NK cell receptors (Ho, W. Y. et al. 2003. Cancer Cell3:431-437; Eshhar, Z. et al. 1993. Proc. Nat. Acad. Sci. USA 90:720-724;Maher, J. and E. T. Davies. 2004. Br. J. Cancer 91:817-821; Zhang, T. etal. 2005. Blood 106:1544-1551).

Additional disclosures generally related to the field of cell-basedtherapies and chimeric NK receptors include WO/2011/05936,WO/2006/036445, U.S. patent application publication no. 2002/0039576,U.S. patent application publication no. 2006/0166314, U.S. provisionalpatent applications No. 61/255,980, filed Oct. 29, 2009, 60/612,836filed Sep. 24, 2004, 60/681,782, filed May 17, 2005, Anderson et al.(2004) Blood 104:1565-1573, and Maeda et al. (2005) Blood 106:749-755,each of which is hereby incorporated by reference herein in itsentirety.

BRIEF SUMMARY

Many human cancer cells naturally express NKp30 ligands. To harness theNKp30 receptor-ligand interaction for cancer therapy, we have createdchimeric NKp30 (chNKp30) receptors by linking human NKp30 to a varietyof other protein domains, including human CD3ζ, CD28, Dap10, CD27, andCD8, that allow for stable protein expression and enhanced or alteredsignal transduction in T cells.

The examples below demonstrate that treatment with chNKp30-expressing Tcells promoted survival and tumor eradication in mice bearing a tumorthat expressed an NKp30 ligand. Additionally, the treatment elicited thehosts to generate memory responses against similar tumors lacking NKp30ligand expression, such that mice that survived the first tumor werealso completely resistant to the tumor re-challenge.

Without intent to be limited by theory, it is believed that these memoryresponses may be mediated by “epitope spreading,” wherein initialtargeting of NKp30 ligands by the chNKp3o-expressing T cells is thoughtto lead to tumor cell death, followed by efficient presentation of tumorantigens by professional APCs (such as DCs), probably as a result of thepresence of proinflammatory cytokines (e.g., IFNγ, TNF-α, and GM-CSF)and chemokines. Cross-priming of host T cells by these APCs may furtherlead to expansion of polyclonal tumor-specific T cells (i.e., epitopespreading). Due to these memory responses, it is predicted that tumorswould be less able to evade immune surveillance through selection oftumor cells bearing mutations or deletions of targeted antigens (Swannet al., J. Clin. Invest. 117: 1137-1146; Kim et al., Immunology 121:1-14). Thus, the methods of the present disclosure can induce polyclonaltumor-specific T cells that will minimize the chances for tumor cells to“escape,” increasing the efficacy of the treatment and reducing thelikelihood of recurrent tumor disease.

More specifically, the examples below demonstrate that chimeric NKp30molecules can be expressed by viral transduction in human T cells andallow these cells to recognize tumor cells that express an NKp30 ligand,B7-H6. The chimeric NKp30 expressing T cells kill these tumor cells andsecrete cytokines (e.g., IFN-γ), but not when cultured withligand-deficient tumor cells.

These chimeric receptors were also expressed in murine T cells and leadto in vitro killing and cytokine release in the presence ofligand-expressing tumor cells. In addition, these murine T cellsexpressing chimeric human NKp30 constructs were demonstrated to removeligand-expressing tumors in vivo and increase survival of mice bearing aligand-expressing lymphoma. Several of these mice become long-termsurvivors. These survivors were resistant to a tumor re-challenge with asimilar, but ligand-deficient, lymphoma. These data show that thischimeric NKp30 receptor T cell treatment can lead to tumor eradicationand suggest induction of long-term tumor immunity in the treatedanimals. Thus, chimeric NKp30 receptors may be a viable therapy for thetreatment of tumors that express NKp30 ligands.

In summary, a chNKp30 receptor can be used to redirect T cells againstNKp30 ligand-expressing tumors. Incorporation of a CD28-signaling domaininto chimeric NKp30 receptors can stimulate both primary andcostimulatory signals for enhanced antitumor activities. NKp30 canrecognize its ligands on several different types of tumor cells, andthis study demonstrates a potential broad therapeutic usefulness of thischNKp30 CAR approach for the treatment of cancer.

We additionally envision a portion of DAP10, or other proteins (such asDAP12) to enhance the function of NKp30. In addition, we have alsocreated chimeric molecules that contain NKp30 fused to more than one ofthese other protein signaling domains. These combinations create newreceptors with novel and unexpected signaling properties, includingcellular cytotoxicity and cytokine production. The properties of thevarious chimeric NKp30 molecules have been empirically determined.

The present disclosure also relates to a method for reducing oreliminating tumors. The method involves introducing into an isolated Tcell of a patient (or an allogeneic T cell, e.g., obtained from acompatible donor) having or suspected of having or at risk fordeveloping a tumor a nucleic acid construct containing a first nucleicacid sequence comprising a promoter operably linked to a second nucleicacid sequence encoding a chimeric receptor protein as described in thepreceding paragraphs, e.g., comprising an NKp30 extracellular domainlinked to a variety of other protein domains, including domains of CD3ζ,CD28, CD8, DAP10, and/or CD27. The T cell is subsequently introducedback into the patient so that the chimeric receptor is expressed on thesurface of the T cell to activate anti-tumor immunity in the patentthereby reducing or eliminating the tumor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a graphical overview of proteins involved in NKp30signaling. Ligand-bound NKp30 can activate signaling through CD3ζ andFcRγ. The long horizontal lines represent the cell membrane, with theextracellular space oriented toward the top of the figure. NKp30interaction with CD3ζ correlates with the NKp30 splice variant isoform,with NKp30 isoform A associating with CD3ζ upon cross-linking, NKp30isoform B constitutively associating with CD3ζ, and NKp30 isoform Cpoorly associating with CD3ζ. ITAM: immunoreceptor tyrosine-basedactivation motif.

FIG. 2 illustrates chimeric NK receptors exemplified herein.Extracellular (EX or EC), transmembrane (TM), and cytoplasmic (CYP)(i.e., intracellular) portions are indicated. Wild-type (WT) andchimeric forms of the receptors are indicated. wtNKp30 indicates themature wild-type NKp30 polypeptide. NKp30-CD3ζ indicates an NKp30cytoplasmic domain fused to the transmembrane and cytoplasmic domains ofCD3ζ. NKp30-CD28-CD3 indicates an NKp30 cytoplasmic domain fused to thetransmembrane and cytoplasmic domains of CD28, and additionally thecytoplasmic domain of CD3ζ. All constructs are human.

FIGS. 3A-I shows the level of surface expression of chimeric NKp30receptors on human T cells for the constructs illustrated in FIG. 2 andadditional constructs. Surface expression was analyzed by flow cytometryusing labeled anti-NKp30 and anti-CD4 mAbs (the latter identifiesCD4-positive T cells). Results are shown for mock-transfected cells(FIG. 3A); wild-type (i.e., non-chimeric) NKp30 transfected cells (FIG.3B); chimeric NKp30-CD3ζ transfected cells (FIG. 3C); chimericNKp30-CD28-CD3ζ transfected cells (FIG. 3D); wild-type (i.e.,non-chimeric) NKp30 transfected cells (FIG. 3E); chimericNKp30-CD28-CD3ζ transfected cells (FIG. 3F); NKp30-CD28(TM)-DAP10-CD3ζtransfected cells (FIG. 3G); NKp30-CD28(TM)-CD3ζ-Dap10 transfected (FIG.3H); and NKp30-CD28(TM)-CD27-CD3ζ (FIG. 3I). The results generallyindicate robust surface expression of the chimeric constructs, exceptthat the level of detected surface expression was somewhat lower for30-tm28-27-z than the other constructs. In contrast, retroviraltransduction of human T cells with wtNKp30 gene did not lead tosignificant surface expression.

FIGS. 3J-O show NKp30 expression on human T cells 7 d after transductionwith NKp30 chimric antigen receptors (CARs). NKp30 expression wasmeasured using the PE-conjugated anti-NKp30 mAbs in combination withanti-CD4-FITC mAbs. More than 99% of cells were CD3+ T cells (data notshown). CD4− T cells are CD8+ T cells. Results are shown formock-transfected cells (FIG. 3J); wild-type (i.e., non-chimeric) NKp30transfected cells (FIG. 3K); chimeric NKp30-CD3ζ transfected cells (FIG.3L); chimeric NKp30-CD8(TM)-CD3ζ transfected cells (FIG. 3M); chimericNKp30-CD28-CD3ζ transfected cells (FIG. 3N) and NKp30-CD28(TM)-CD3ζtransfected cells (FIG. 3O). The data are representative of threeexperiments.

FIGS. 4A-B. Expression of NKp30 ligands on tumor cells and PBMC. Humantumor cell lines as well as human PBMCs were screened for the expressionof NKp30 ligand mRNAs by RT-PCR using primers specific for the NKp30ligands BAT3 and B7-H6, as well as a housekeeping gene (GAPDH) as aninternal positive control. All tested human tumor cells had detectableamounts of BAT3 mRNA, whereas B7-H6 expression was readily detected inHeLa, U937, Panc-1, T47D, RPM18226, K562, and A375 cells, but expressionwas at lower levels or undetectable in MCF-7, DU145, IM9, U266 and humanPBMCs.

FIG. 5A. Surface expression of NKp30 ligands on tumor cells. K562, A375and HeLa cells express high amounts of NKp30 ligands, whereas U937,RPMP8226, T47D and Panc-1 cells express marginal levels of NKp30ligands. Some tumor cells (IM9, MM.1s, MCF-7 and DU145) as well as humanPBMCs do not express NKp30 ligands. B7-H6 mRNA amounts are correlatedwith surface expression of NKp30 ligands, suggesting that B7-H6 is themajor surface ligand of NKp30 in the tested tumors.

FIG. 5B. NKp30 ligand expression on the surface of human tumor celllines was measured by flow cytometry using anti-B7-H6 mAbs (solid line)or a soluble NKp30 receptor fused to a mouse IgG2a Fc region(NKp30-mIgG2a; dashed line), followed by staining withallophycocyanin-conjugated goat anti-mouse IgG. Isotype controls areshown as a dotted line.

FIGS. 6A-E illustrates production of IFN-γ after co-culture oftransfected T cells with NKp30 ligand-positive cells but not withligand-negative cells (or T cells alone). FIGS. 6A-B show thatNKp30-CD3ζ+ (grey bars, middle bar in each group) and NKp30-CD28-CD3ζ+ T(black bars, rightmost bar in each group) cells produced significantamounts of IFN-γ, indicating that these chimeric NKp30-modified T cellscould functionally recognize NKp30 ligand-bearing tumor cells. Incontrast, wild-type NKp30-modified T cells (FIG. 6A, white bars, leftbar in each group) did not show any significant response to thestimulation by NKp30-ligand positive cells. FIG. 6C illustrates IFN-γproduction after co-culture with T cells that were mock-transfected,transfected with wild-type NKp30, NKp30-CD28-CD3ζ,NKp30-CD28(TM)-DAP10-CD3ζ, NKp30-CD28(TM)-CD3ζ-Dap10, andNKp30-CD28(TM)-CD27-CD3ζ (leftmost through rightmost bars in each group,respectively). IFN-γ production was highest in the cultures containingNKp30-CD28-CD3ζ transfected T cells. FIGS. 6D-E further illustrate thatNKp30 chimeric antigen receptor-modified T cells respond to NKp30ligand-positive cells by producing IFN-γ. Five to seven days afterretroviral transduction, NKp30 chimeric antigen receptor-modified Tcells (100,000 cells) were cocultured with either irradiated ormitomycin C-treated tumor cells for 24 h. Suspension tumor cells(100,000 cells) (D) and adherent tumor cells (25,000 cells) (F) wereused. RMA and MCF-7 cells were used as negative controls. IFN-γ amountsin the supernatants were analyzed with ELISA. Results are shown asmean+/−SD. Asterisks (*) indicate p<0.05.

FIGS. 7A-B. Chimeric NKp30-bearing human T cells lysed NKp30-ligandpositive tumor cells. Effector T cells derived from human PBMCs weremodified with wtNKp30, NKp30-3ζ, or NKp30-CD28-3ζ and cocultured withtumor cells at a ratio of 5:1 in 5-h LDH-release assays. In FIG. 7A,NKp30-CD3ζ+ (black bars, middle bar in each group) or NKp30-CD28-CD3ζ+(diagonal hatched bars, rightmost bar in each group) T cells lysed NKp30ligand-positive cells (RMA/B7-H6, K562, U937, HeLa, Panc-1, A375, andT47D) but not ligand-negative cells (cell line RMA) indicating thatthese chimeric NKp30-modified T cells could functionally recognize NKp30ligand-bearing tumor cells in a specific manner. In contrast, a farlower percentage of tumor cells were lysed in the presence of wild-typeNKp30-modified T cells (FIG. 7A, light gray bars, left bar in eachgroup). NK30-CD28-CD3ζ+ killed a greater percentage of NKp30ligand-positive tumor cells than NKp30-CD3ζ. Results are shown fortriplicate experiments (mean+/−SD). Similarly, in FIG. 7B, the T cellsexpressing the 30-28-z, 30-tm28-10-z, 30-tm28-z-10, and 30-tm28-27-zconstructs lysed NKp30 ligand-positive cells (RMA-B7H6 and K562) but notthe negative control RMA cell (which lacks B7H6 expression), whereas farfewer tumor cells were lysed in the presence of T cells expressingwild-type NKp30. FIG. 7B legend: constructs in each group of bars, inorder from left to right, were: NKp30, 30-28-z, 30-tm28-10-z,30-tm28-z-10, and 30-tm28-27-z.

FIG. 7C further illustrates that lysis was mediated by the chimericNKp30 constructs. NKp30-mIgG2a significantly reduced NKp30-28-3ζ-bearingT cell-mediated cytotoxicity. These results demonstrated that chimericNKp30-bearing T cells killed ligand-positive tumor cells, and theinteraction between chimeric NKp30 receptors and NKp30 ligands wasessential for chimeric NKp30-mediated T cell function. Effector T cellsmodified with wtNKp30 or NKp30-CD28-3ζ were cocultured with target cellsK562 in the presence of 10 μg/ml NKp30-mIgG2a or mouse IgG at a ratio of5:1; the percentage of specific lysis was determined after a 5-hLDH-release assay. The data are presented as mean+/−SD and arerepresentative of two independent experiments.

FIG. 8. Shows data indicating that PI3 kinase is involved inNKp30-CD28-CD3ζ-mediated cytotoxicity. Specific lysis was significantlydecreased for the chimeric NKp30-CD28-CD3ζ T cells incubated with theLy294002 inhibitor, indicating that the PI3 kinase plays a role inNKp30-CD28-CD3ζ-mediated cytotoxicity. NKp30-modified effector T cellswere incubated with a PI3K inhibitor LY294002 (10 μM) at 37° C. for 1 hbefore coculture with K562 target cells at a E:T ratio of 5:1 in 5-hLDH-release assays. Vehicle controls are 0.1% DMSO. The data shown arethe mean+/−SD of triplicates and are representative of two independentexperiments. Asterisk (*) indicates p<0.05.

FIGS. 9A-D. NKp30 expression on mouse T cells. Expression levels areshown for mock-transfected cells (FIG. 9A); wild-type (i.e.,non-chimeric) NKp30 transfected cells (FIG. 9B); chimeric NKp30-CD3ζtransfected cells (FIG. 9C); and chimeric NKp30-CD28-CD3ζ transfectedcells (FIG. 9D).

FIGS. 9E-J further exemplifies chimeric human NKp30 expression on mousecells. Human NKp30 expression on mouse T cells 7 d after transduction.NKp30 expression was detected using the PE-conjugated anti-NKp30 mAb incombination with the anti-mouse CD4-FITC mAb. CD42 T cells are CD8+ Tcells. The data are representative of three experiments. Expressionlevels are shown for mock-transfected cells (FIG. 9E); wild-type (i.e.,non-chimeric) NKp30 transfected cells (FIG. 9F); chimeric NKp30-CD3ζtransfected cells (FIG. 9G); chimeric NKp30-CD8(TM)-CD3ζ transfectedcells (FIG. 9H); chimeric NKp30-CD28(TM)-CD3ζ transfected cells (FIG.9I), and chimeric NKp30-CD28-CD3ζ transfected cells (FIG. 9J).

FIGS. 10A-B. Human NKp30 receptors are functional in mouse T cells.Effector T cells derived from B6 (open), perforin-deficient (Pfp−/−,filled) mice that were modified with NKp30 receptors were co-culturedwith RMA or RMA/B7-H6 cells at an E:T ratio of 1:1 in 5-h LDH-releaseassays. The T cells lysed a significantly higher percentage of NKp30ligand-positive cells (RMA/B7-H6, FIG. 10B) than ligand-negative cells(cell line RMA, FIG. 10A). The data are presented as mean±SD oftriplicates and are representative results from two independentexperiments (FIGS. 10A and 10B). Specific lysis was substantiallydecreased with the Pfp−/− cells.

FIG. 10C shows amounts of IFN-γ produced by transduced murine T cells inresponse to NKp30 ligand-positive cells. Seven days after retroviraltransduction, NKp30-modified T cells (100,000 cells) were coculturedwith irradiated RMA/B7-H6 cells (100,000 cells) for 24 h. Mouse lymphomacell line RMA was used as a negative control. IFN-γ amounts in thesupernatants were analyzed by ELISA. Results are shown as mean+/−SD.

FIGS. 11A-C. Integration of CD28 TM and signaling domains into chimericNKp30 receptor leads to long-term survival in tumor-bearing mice treatedwith these T cells in vivo. Mouse T cells transduced with chimeric NKp30enhanced survival of mice injected with lymphoma cells, specificallyRMA/B7-H6 that express the NKp30 ligand B7-H6 (schatically illustratedin FIG. 11A). Integration of CD28 TM and signaling domains into chimericNKp30 receptor was demonstrated to lead to better anti-tumor in vivoefficacy. RMA/B7-H6 cells (105) were administered i.v. on day 0. On day5, 7 and 9, tumor-bearing mice were injected with T cells (5×10⁶) thatwere modified to express wtNKp30 (♦), NKp30-CD3ζ (▴), NKp30-CD8-CD3ζ (●)and NKp30-CD28-CD3ζ (□), respectively (FIG. 11B). Injection with asaline solution (HBSS) was used as negative control. Data are presentedin Kaplan-Meier survival curves. Additionally, surviving mice gainedligand-independent tumor resistance. Long-term survivors werere-challenged with a similar, but ligand-deficient, lymphoma,specifically RMA cells that had not been transformed with the B7-H6construct. Overall survival was determined, and none of there-challenged mice had tumor growth by the end of the study period,whereas naïve mice did (FIG. 11C).

FIGS. 12A-B. Human dendritic cells (“DCs”) bind to NKp30 and canstimulate autologous NKp30-CD28-3ζ-modified T cells to produce IFN-γ.(A) The cell surface phenotype and binding to NKp30 of PBMC-derivedhuman DCs (both iDCs and mDCs) was determined by flow cytometry.Specific mAb or NKp30-Ig as indicated (solid line) or an isotype controlAb staining (dashed line) is shown. (B) Five to seven days afterretroviral transduction, NKp30 chimeric antigen receptor-modified Tcells (10⁵ cells) were cocultured with either iDCs or mDCs at a ratio of5:1 (T/DC) for 24 h. IFN-γ amounts in the supernatants were determinedby ELISA. Results shown (mean+SD) are representative of two experiments.Asterisk (*) indicates p<0.05. As further discussed infra, because NKp30ligands can be expressed by human dendritic cells, these results helpconfirm that the methods and compositions of the present disclosure maybe useful to prevent or treat diseases where elimination of dendriticcells may be helpful, such as autoimmune diseases or rejection oftransplanted organs.

FIGS. 13A-B. Engagement of NKp30-CD28-3ζ receptor led to increased Tcell proliferation and upregulation of IL-2 and Bcl-x_(L). (A) NKp30receptor (either wtNKp30 or chimeric NKp30)-modified human T cells werelabeled with CFSE, as described in the examples below, and coculturedwith HeLa cells (100,000 cells, NKp30 ligand-positive) in the presenceof a small amount of IL-2 (25 U/ml) for 3 d. Analysis of T cellproliferation (i.e., CFSE dilution) was performed on both NKp30+ (FL4)and NKp30-cells within the same mixed T cell population by flowcytometry. (B) NKp30-modified T cells (250,000 cells) were cultured inanti-NKp30 mAb (4 μg)-coated 24-well plates for 24 h. Mouse IgG was usedas a negative control. IL-2 gene expression was determined by real-timePCR, as described in the examples below. Results are shown as foldincrease, in which the IL-2 gene expression in the control mAb-treated Tcells was normalized to 1. Data are presented as mean+/−SD from twoindependent experiments. (C) Twenty-four hours after cross-linking withimmobilized anti-NKp30 mAbs, as described in the examples below, T cellswere collected. Bcl-x_(L) expression was determined by flow cytometryafter intracellular staining with anti-Bcl-x_(L)-FITC (solid line) orisotype control mAbs (dashed line). Asterisk (*) indicates p<0.05.

FIG. 14 schematically illustrates additional exemplary chimeric NKp30receptor constructs. The two horizontal lines represent a cell membrane,with the portion above both lines corresponding to the extracellulardomain (NKp30 extracellular domain in each Illustrated construct), theportion between the horizontal lines corresponding to the transmembranedomain (e.g., CD3ζ transmembrane domain or CD28 domain in theillustrated construct, though other transmembrane domains are also usedin exemplary embodiments, e.g., a CD8 transmembrane domain in theconstruct shown in FIG. 19); and the portion below both horizontal linesincluding the one or more activation domain(s) (e.g., the cytoplasmicdomains of CD3ζ, CD28, DAP10, CD27, and combinations thereof.Illustrated constructs include wild-type NKp30 (Wt-NKp30); NKp30-CD3ζ,containing the NKp30 extracellular domain and CD3ζ transmembrane andcytoplasmic domains; 30-tm28-3ζ (also referred to herein asNKp30-CD28(TM)-CD3ζ); 30-28-3ζ (also referred to herein asNKp30-CD28-CD3ζ); 30-tm28-10-3ζ (also referred to herein asNKp30-CD28(TM)-DAP10-CD3ζ); 30-tm28-3ζ-10 (also referred to herein asNKp30-CD28(TM)-CD3ζ-Dap10); and 30-tm28-27-3ζ (also referred to hereinas NKp3-CD28(TM)-CD27-CD3ζ).

FIG. 15 provides a polypeptide sequence of a wild-type human NKp30 andillustrates domains thereof.

FIGS. 16-22 provide polypeptide sequences of chimeric NKp30 receptors,and exemplary polynucleotide sequences are shown below. In thesefigures, the labels “TM” or “TM domain” refer to transmembrane domainsequences; the labels “EC” or “EC domain” refer to extracellularsequences (e.g., NKp30 ligand-binding domain sequences) and “CYP” or“CYP domain” refer to cytoplasmic domain sequences (which includesignaling domains of the identified polypeptides). The identifiedpolypeptides refer to the wild-type sequences from which the respectivedomains were derived.

FIG. 16 provides the polypeptide sequence of an NKp30-CD3ζ (alsoreferred to herein as NKp30-CD3ζ or NKp30-C or NKp30-3z) constructcomprising a signal peptide and extracellular domain of NKp30, and atransmembrane and cytoplasmic domain of CD3ζ. An exemplary codingsequence of this NKp30-CD3ζ construct is:

(SEQ ID NO: 124) ATGGCCTGGATGCTGTTGCTCATCTTGATCATGGTCCATCCAGGATCCTGTGCTCTCTGGGTGTCCCAGCCCCCTGAGATTCGTACCCTGGAAGGATCCTCTGCCTTCCTGCCCTGCTCCTTCAATGCCAGCCAAGGGAGACTGGCCATTGGCTCCGTCACGTGGTTCCGAGATGAGGTGGTTCCAGGGAAGGAGGTGAGGAATGGAACCCCAGAGTTCAGGGGCCGCCTGGCCCCACTTGCTTCTTCCCGTTTCCTCCATGACCACCAGGCTGAGCTGCACATCCGGGACGTGCGAGGCCATGACGCCAGCATCTACGTGTGCAGAGTGGAGGTGCTGGGCCTTGGTGTCGGGACAGGGAATGGGACTCGGCTGGTGGTGGAGAAAGAACATCCTCAGCTAGGGGCTAGCCTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGC CCTGCCCCCTCGC.

FIG. 17 provides the polypeptide sequence of an NKp30-CD28-CD3ζ (alsoreferred to herein as NKp30-CD28-CD3, 30-28-z, 30-28-3ζ,NKp30-CD28-CD3z, or NKp30-CD28-ζ) construct comprising a signal peptideand extracellular domain of NKp30, a transmembrane and cytoplasmicdomain of CD28, and a further cytoplasmic domain of CD3ζ. An exemplarycoding sequence of this NKp30-CD28-CD3ζ construct is:

(SEQ ID NO: 125) ATGGCCTGGATGCTGTTGCTCATCTTGATCATGGTCCATCCAGGATCCTGTGCTCTCTGGGTGTCCCAGCCCCCTGAGATTCGTACCCTGGAAGGATCCTCTGCCTTCCTGCCCTGCTCCTTCAATGCCAGCCAAGGGAGACTGGCCATTGGCTCCGTCACGTGGTTCCGAGATGAGGTGGTTCCAGGGAAGGAGGTGAGGAATGGAACCCCAGAGTTCAGGGGCCGCCTGGCCCCACTTGCTTCTTCCCGTTTCCTCCATGACCACCAGGCTGAGCTGCACATCCGGGACGTGCGAGGCCATGACGCCAGCATCTACGTGTGCAGAGTGGAGGTGCTGGGCCTTGGTGTCGGGACAGGGAATGGGACTCGGCTGGTGGTGGAGAAAGAACATCCTCAGCTAGGGGCTAGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAAGCTTAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGC CCCCTCGC.

FIG. 18 provides the polypeptide sequence of an NKp30-CD28(TM)-CD3ζ(also referred to herein as NKp30-CD28(TM)-ζ or 30-tm28-3) constructcomprising a signal peptide and extracellular domain of NKp30, and atransmembrane and cytoplasmic domain of CD3ζ. An exemplary codingsequence of this NKp30-CD28(TM)-CD3ζ construct is:

(SEQ ID NO: 126) ATGGCCTGGATGCTGTTGCTCATCTTGATCATGGTCCATCCAGGATCCTGTGCTCTCTGGGTGTCCCAGCCCCCTGAGATTCGTACCCTGGAAGGATCCTCTGCCTTCCTGCCCTGCTCCTTCAATGCCAGCCAAGGGAGACTGGCCATTGGCTCCGTCACGTGGTTCCGAGATGAGGTGGTTCCAGGGAAGGAGGTGAGGAATGGAACCCCAGAGTTCAGGGGCCGCCTGGCCCCACTTGCTTCTTCCCGTTTCCTCCATGACCACCAGGCTGAGCTGCACATCCGGGACGTGCGAGGCCATGACGCCAGCATCTACGTGTGCAGAGTGGAGGTGCTGGGCCTTGGTGTCGGGACAGGGAATGGGACTCGGCTGGTGGTGGAGAAAGAACATCCTCAGCTAGGGGCTAGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAAGCTTAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.

FIG. 19 provides the polypeptide sequence of an NKp30-CD8-CD3ζ (alsoreferred to herein as NKp30-CD8-ζ or NKp30-CD8(TM)-3ζ) constructcomprising a signal peptide and extracellular domain of NKp30, atransmembrane domain of CD8, and a cytoplasmic domain of CD3ζ. Anexemplary coding sequence of this NKp30-CD8-CD3ζ construct is:

(SEQ ID NO: 127) ATGGCCTGGATGCTGTTGCTCATCTTGATCATGGTCCATCCAGGATCCTGTGCTCTCTGGGTGTCCCAGCCCCCTGAGATTCGTACCCTGGAAGGATCCTCTGCCTTCCTGCCCTGCTCCTTCAATGCCAGCCAAGGGAGACTGGCCATTGGCTCCGTCACGTGGTTCCGAGATGAGGTGGTTCCAGGGAAGGAGGTGAGGAATGGAACCCCAGAGTTCAGGGGCCGCCTGGCCCCACTTGCTTCTTCCCGTTTCCTCCATGACCACCAGGCTGAGCTGCACATCCGGGACGTGCGAGGCCATGACGCCAGCATCTACGTGTGCAGAGTGGAGGTGCTGGGCCTTGGTGTCGGGACAGGGAATGGGACTCGGCTGGTGGTGGAGAAAGAACATCCTCAGCTAGGGGCTAGCATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCAAGCTTAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCA GGCCCTGCCCCCTCGCTAA.

FIG. 20 provides the polypeptide sequence of a NKp30-CD28(TM)-DAP0-CD3ζ(also referred to herein as 30-tm28-10-3z, Dap10a, 30-tm28-10-7, or30-tm2-10-3ζ) construct comprising a signal peptide and extracellulardomain of NKp30, transmembrane domain of CD28, cytoplasmic domain ofDap10, and a cytoplasmic domain of CD3ζ. An exemplary coding sequence ofthis NKp30-CD28(TM)-DAP10-CD3ζ construct is:

(SEQ ID NO: 64) ATGGCCTGGATGCTGTTGCTCATCTTGATCATGGTCCATCCAGGATCCTGTGCTCTCTGGGTGTCCCAGCCCCCTGAGATTCGTACCCTGGAAGGATCCTCTGCCTTCCTGCCCTGCTCCTTCAATGCCAGCCAAGGGAGACTGGCCATTGGCTCCGTCACGTGGTTCCGAGATGAGGTGGTTCCAGGGAAGGAGGTGAGGAATGGAACCCCAGAGTTCAGGGGCCGCCTGGCCCCACTTGCTTCTTCCCGTTTCCTCCATGACCACCAGGCTGAGCTGCACATCCGGGACGTGCGAGGCCATGACGCCAGCATCTACGTGTGCAGAGTGGAGGTGCTGGGCCTTGGTGTCGGGACAGGGAATGGGACTCGGCTGGTGGTGGAGAAAGAACATCCTCAGCTAGGGGCTAGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCCTGTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAAGCTTAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.

FIG. 21 provides the polypeptide sequence of a NKp30-CD28(TM)-CD3ζ-Dap10(also referred to herein as 30-tm28-3z-10, Dap10b, 30-tm28-z-10, or30-tm28-3ζ-10) construct construct comprising a signal peptide andextracellular domain of NKp30, transmembrane domain of CD28, acytoplasmic domain of CD3ζ, and a cytoplasmic domain of Dap10. Anexemplary coding sequence of this NKp30-CD28(TM)-CD3ζ-Dap10 constructis:

(SEQ ID NO: 66) ATGGCCTGGATGCTGTTGCTCATCTTGATCATGGTCCATCCAGGATCCTGTGCTCTCTGGGTGTCCCAGCCCCCTGAGATTCGTACCCTGGAAGGATCCTCTGCCTTCCTGCCCTGCTCCTTCAATGCCAGCCAAGGGAGACTGGCCATTGGCTCCGTCACGTGGTTCCGAGATGAGGTGGTTCCAGGGAAGGAGGTGAGGAATGGAACCCCAGAGTTCAGGGGCCGCCTGGCCCCACTTGCTTCTTCCCGTTTCCTCCATGACCACCAGGCTGAGCTGCACATCCGGGACGTGCGAGGCCATGACGCCAGCATCTACGTGTGCAGAGTGGAGGTGCTGGGCCTTGGTGTCGGGACAGGGAATGGGACTCGGCTGGTGGTGGAGAAAGAACATCCTCAGCTAGGGGCTAGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAAGCTTAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCTGTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGC.

FIG. 22 provides the polypeptide sequence of a NKp3-CD(TM)-CD27-CD3ζ(also referred to herein as 30-tm28-27-3z, 30-tm28-27-z, or30-tm28-27-3ζ) construct comprising a signal peptide and extracellulardomain of NKp30, transmembrane domain of CD28, a cytoplasmic domain ofCD27, and a cytoplasmic domain of CD3ζ. An exemplary coding sequence ofthis NKp30-CD28(TM)-CD27-construct is:

(SEQ ID NO: 68) ATGGCCTGGATGCTGTTGCTCATCTTGATCATGGTCCATCCAGGATCCTGTGCTCTCTGGGTGTCCCAGCCCCCTGAGATTCGTACCCTGGAAGGATCCTCTGCCTTCCTGCCCTGCTCCTTCAATGCCAGCCAAGGGAGACTGGCCATTGGCTCCGTCACGTGGTTCCGAGATGAGGTGGTTCCAGGGAAGGAGGTGAGGAATGGAACCCCAGAGTTCAGGGGCCGCCTGGCCCCACTTGCTTCTTCCCGTTTCCTCCATGACCACCAGGCTGAGCTGCACATCCGGGACGTGCGAGGCCATGACGCCAGCATCTACGTGTGCAGAGTGGAGGTGCTGGGCCTTGGTGTCGGGACAGGGAATGGGACTCGGCTGGTGGTGGAGAAAGAACATCCTCAGCTAGGGGCTAGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCCTCGAGCAACGAAGGAAATATAGATCAAACAAAGGAGAAAGTCCTGTGGAGCCTGCAGAGCCTTGTCGTTACAGCTGCCCCAGGGAGGAGGAGGGCAGCACCATCCCCATCCAGGAGGATTACCGAAAACCGGAGCCTGCCTGCTCCCCCAAGCTTAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

NKp30 is a natural cytotoxicity receptor that is expressed on NK cellsand recognizes B7-H6, which is expressed on several types of tumors butfew normal cells. To target effector T cells against B7-H6+ tumors, wedeveloped several chimeric antigen receptors based on NKp30, includingreceptors that contain the CD28- and/or CD3γ-signaling domains with thetransmembrane domains from CD3γ, CD28, or CD8α. ChimericNKp30-expressing T cells responded to B7-H6+ tumor cells. T cellsexpressing NKp30 chimeric antigen receptors (CARs) produced IFN-γ andkilled B7-H6 ligand-expressing tumor cells; this response was dependentupon ligand expression on target cells but not on MHC expression.PBMC-derived dendritic cells also express NKp30 ligands, includingimmature dendritic cells, and they stimulated NKp30 CAR-bearing T cellsto produce IFN-γ, but to a lesser extent. The addition of aCD28-signaling domain significantly enhanced the activity of the NKp30CAR in a PI3K-dependent manner.

Adoptive transfer of T cells expressing a chimeric NKp30 receptorcontaining a CD28-signaling domain inhibited the growth of aB7-H6-expressing murine lymphoma (RMA/B7-H6) in vivo. Moreover, micethat remained tumor-free were resistant to a subsequent challenge withthe wild-type RMA tumor cells, suggesting the generation of immunityagainst other tumor antigens. These results demonstrates the specificityand therapeutic potential of adoptive immunotherapy with NKp30 chimericantigen receptor-expressing T cells against B7-H6+ tumor cells in vivo.

The present disclosure relates to a chimeric receptor moleculecomprising an NKp30 extracellular domain expressed on the surface of a Tcell to activate killing of a tumor cell. Nucleic acid sequencesencoding the chimeric receptor molecule are introduced into a patient'sT-cells (or compatible T cells, e.g., an allogeneic T cell obtained froma compatible donor or a cell bank) and T-cells that express the chimericreceptor molecule are subsequently introduced into the patient. In thismanner, the chimeric receptor molecules provide a means for thepatient's own immune cells to recognize and destroy tumor cells,activate anti-tumor immunity, and establish long-term, specific,anti-tumor responses for treating tumors or preventing re-growth ofdormant or residual tumor cells.

By way of illustration, human chimeric receptor molecules composed of anNKp30 extracellular domain in combination with transmembrane and/orcytoplasmic domains of CD3ζ and/or CD28 were generated and expressed inhuman T-cells. Specifically, a gene encoding a chimeric receptorcomprising the extracellular domain of human NKp30 and transmembrane andcytoplasmic domains of human CD3ζ (NKp30-CD3ζ receptor) was generated. Agene encoding a chimeric receptor comprising the extracellular domain ofhuman NKp30, the transmembrane domain of CD28, and cytoplasmic domain ofhuman CD3ζ (NKp30-CD28-CD3ζ receptor) was also generated. As describedfurther in the examples below, these chimeric NKp30 genes wereintroduced into human T cells ex vivo by retroviral transduction, andwere shown to be efficiently surface-expressed. Moreover, T cellsexpressing these chimeric NKp30 receptors (“chimeric NKp30 T cells”)were demonstrated to be specifically activated by and lysed NKp30ligand-positive tumor cells. The same (human) chimeric NKp30 genes wereintroduced into mouse T cells ex vivo by retroviral transduction andwere demonstrated to be surface-expressed by mouse cells as well, andtransduced mouse cells were specifically activated by and lysed NKp30ligand-positive tumor cells. Moreover, the transduced mouse T cellsincreased survival of mice injected with NKp30-ligand expressing tumorcells. Several mice became long-term survivors that were resistant to atumor re-challenge with a similar, but NKp30 ligand-deficient, lymphoma.These results indicate that this chimeric T cell treatment can lead totumor eradication and suggest induction of long-term tumor immunity inthe animals.

Definitions

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

“Allogeneic T cell” refers to a T cell from a donor having a tissue thatis not genetically identical to the recipient. The T cell may have anHLA type that partially or fully matches the recipient or does not matchthe recipient. In some instances allogeneic transplant donors may berelated (usually a closely HLA matched sibling), syngeneic (amonozygotic ‘identical’ twin of the patient) or unrelated (donor who isnot related and found to have very close degree of HLA matching). TheHLA genes fall in two categories (Type I and Type I). In general,mismatches of the Type-I genes (i.e. HLA-A, HLA-B, or HLA-C) increasethe risk of graft rejection. A mismatch of an HLA Type II gene (i.e.HLA-DR, or HLA-DQB1) increases the risk of graft-versus-host disease.

In the context of the present disclosure, a T cell progenitor refers toany cell that may give rise to a T cell including but not limited toadult and embryonic stem cells, induced stem cells, hematopoietic stemcells (e.g., CD34+ hematopoietic stem cells), thymocytes, and Tlymphocyte-restricted progenitors typically found in the thymus.Additional exemplary T cell progenitors include thymocytes in the doublenegative stages (each negative for both CD4 and CD8), such as the doublenegative 1 stage (Lineage-CD44+CD25−CD117+), double negative 2 stage(Lineage-CD44+CD25+CD117+), double negative 3 stage(Lineage-CD44−CD25+), double negative 4 stage (Lineage-CD44−CD25−),double positive stage (CD4+CD8+) and/or single positive stage (CD4+CD8−or CD4−CD8+).

In the context of the present disclosure, a “bank of tissue matchedchimeric NKp30 T cells” refers to different compositions each containingT cells of a specific HLA allotype which express a chimeric NKp30receptor according to the present disclosure. Ideally this bank willcomprise compositions containing T cells of a wide range of differentHLA types that are representative of the human population. Such a bankof engineered chimeric NKp30 T cells will be useful as it willfacilitate the availability of T cells suitable for use in differentrecipients, such as cancer patients.

As used herein. CD3ζ (CD3ζ) refers to human polypeptide orpolynucleotide (or orthologs in other species if the context soindicates) having exemplary sequences as follows. Two CD3ζ transcriptvariants have been reported. One transcript variant, CD3ζ transcriptvariant 1, has Genbank accession number NM_198053 (polynucleotide),encoding the polypeptide CD3ζ chain isoform 1 precursor (NP_0.932170.1)having the sequence:MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 1) which isannotated as comprising a signal peptide (residues 1-21, i.e.,MKWKALFTAAILQAQLPITEA (SEQ ID NO: 2)) and a mature peptide (residues22-164, i.e.,QSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 3)), and is further annotated ascomprising a transmembrane region (residues 31-51, i.e.,LCYLLDGILFIYGVILTALFL (SEQ ID NO: 4)) and additionally includes thecytoplasmic domain sequenceRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 5).

A second transcript variant, CD3ζ transcript variant 2, has Genbankaccession number NM_000734.3 (polynucleotide), encoding the polypeptideCD3ζ chain isoform 2 precursor (NP_000725.1) having the sequence:MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 6) which isannotated as comprising a signal peptide (residues 1-21, i.e.,MKWKALFTAAILQAQ (SEQ ID NO: 7)) and a mature peptide (residues 22-164,i.e., LPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP (SEQ ID NO: 8)), and is further annotatedas comprising a transmembrane region (residues 31-51, i.e.,LCYLLDGILFIYGVILTALFL (SEQ ID NO: 9)) and additionally includes thecytoplasmic domain sequenceRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 10).

As used herein, NKp30 refers to human polypeptide or polynucleotide (ororthologs in other species if the context so indicates) having exemplarysequences as follows. Three NKp30 isoforms have been reported. A humanNKp30 isoform A gene has Genbank accession number NP_667341.1 having thesequence: MAWMLLLILIMVHPGSCALWVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLGAGTVLLLRAGFYAVSFLSVAVGSTVYYQGKCLTWKGPRRQLPAVVPAPLPPPCGSSAHLLPPVPGG (SEQ ID NO: 11) which is annotated ascomprising a transmembrane region (residues 136-156, i.e.,AGTVLLLRAGFYAVSFLSVAV (SEQ ID NO: 12)) and includes a cytoplasmic domainhaving the sequence GSTVYYQGKCLTWKGPRRQLPAVVPAPLPPPCGSSAHLLPPVPGG (SEQID NO: 13)) and additionally includes the signal peptide sequenceMAWMLLLILIMVHPGSCA (SEQ ID NO: 14) and an extracellular domain havingthe sequenceLWVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLG (SEQ ID NO:15).

A second transcript variant, human NKp30 isoform B gene has Genbankaccession number NP_001138938.1 having the sequence:MAWMLLLILIMVHPGSCALWVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLGAGTVLLLRAGFYAVSFLSVAVGSTVYYQGKYAKSTLSGFPQL (SEQ ID NO:16) which is annotated as comprising the same transmembrane regionsequence as isoform A (residues 136-156, i.e., AGTVLLLRAGFYAVSFLSVAV(SEQ ID NO: 17)) and includes the signal peptide sequenceMAWMLLLILIMVHPGSCA (SEQ ID NO: 18), extracellular domain sequenceLWVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLG (SEQ ID NO:19), and cytoplasmic domain sequence GSTVYYQGKYAKSTLSGFPQL (SEQ ID NO:20).

A third transcript variant, human NKp30 isoform C gene has Genbankaccession number NP_001138939.1 having the sequence:MAWMLLLILIMVHPGSCALWVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLGAGTVLLLRAGFYAVSFLSVAVGSTVYYQGKCHCHMGTHCHS SDGPRGVIPEPRCP(SEQ ID NO: 21) which contains the same sequence region sequence asisoforms A and B which is likewise expected to function as atransmembrane sequence (residues 136-156, i.e., AGTVLLLRAGFYAVSFLSVAV(SEQ ID NO: 22)) and includes the signal peptide sequenceMAWMLLLILIMVHPGSCA (SEQ ID NO: 23), extracellular domain sequenceLWVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLG(S EQ ID NO:24) and cytoplasmic domain sequence GSTVYYQGKCHCHMGTHCHSSDGPRGVIPEPRCP(SEQ ID NO: 25).

As used herein, CD28 refers to human polypeptide or polynucleotide (ororthologs in other species if the context so indicates) having exemplarysequences as follows. Three human CD28 transcript variants have beenreported. One transcript variant, human CD28 isoform 1, has Genbankaccession number AF222341_1 having the sequence:

(SEQ ID NO: 26) MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSWKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS.

A second transcript variant, human CD28 isoform 2, has Genbank accessionnumber AAF33793.1 having the sequence:

(SEQ ID NO: 27) MLRLLLALNLFPSIQVTGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYR S.

CD28 isoforms 2 and 3 each include the transmembrane sequenceFWVLVVVGGVLACYSLLVTVAFIIFWVRSK (SEQ ID NO: 28) and cytoplasmic domainsequence RSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 29). Inexemplary embodiments, the transmembrane sequence may include adjacentsequences annotated as part of another domain, e.g., an exemplarytransmembrane sequence is FWVLVVVGGVLACYSLLVTVAFIFWVRSKRS (SEQ ID NO:30).

A third transcript variant, human CD28 isoform 3, has Genbank accessionnumber AAF33794.1 having the sequence:

(SEQ ID NO: 31) MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSYNEKSNGTIIHVKGKHLCPSPLFPGPSKPYAPPRDFAAYRS.

As used herein, CD8 refers to human polypeptide or polynucleotide (ororthologs in other species if the context so indicates) having exemplarysequences as follows. Two human CD8 transcript variants have beenreported. One transcript variant, CD8α chain isoform 1 precursor, hasGenbank accession number NP_001759.3 having the sequence:

(SEQ ID NO: 32) MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV

which is annotated as comprising a signal peptide (residues 1-21, i.e.,MALPVTALLLPLALLLHAARP (SEQ ID NO: 33)) and a mature peptide (residues22-235, i.e.,SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKFTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO: 34)), and is furtherannotated as comprising a transmembrane region (residues 183-203, i.e.,IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 35)).

A second transcript variant, CD8α chain isoform 2 precursor, has Genbankaccession number NP_741969.1 has the sequence

(SEQ ID NO: 36) MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAGNRRRVCKCPRPVVKSGDKPSLSAR YV

which contains the same sequence annotated as a signal peptide inisoform 1 (residues 1-21) which is likewise expected to function as asignal peptide, with the remaining residues (i.e., residues 22-198)likewise expected to correspond to the mature polypeptide.

As used herein, DAP10 refers to human polypeptide or polynucleotide (ororthologs in other species if the context so indicates) having exemplarysequences as follows. Two human DAP10 transcript variants have beenreported. One transcript variant, human DAP10 isoform 1 precursor(hematopoietic cell signal transducer isoform 1 precursor) has Genbankaccession number NP_055081.1 having the sequence:

(SEQ ID NO: 37) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRG

which is annotated as comprising a signal peptide (residues 1-19, i.e.,MIHLGHILFLLLLPVAAAQ (SEQ ID NO: 38)) and a mature peptide (residues20-93, i.e.,TTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO: 39)), and is further annotated as comprisinga transmembrane region (residues 49-69, i.e., LLAGLVAADAVASLLIVGAVF (SEQID NO: 40)) and additionally includes the cytoplasmic domain sequence

(SEQ ID NO: 41) LCARPRRSPAQEDGKVYINMPGRG.

A second transcript variant, DAP10 isoform 2 precursor (hematopoieticcell signal transducer isoform 2 precursor) has Genbank accession numberNP_001007470.1 having the sequence:

(SEQ ID NO: 42) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQDGKVYINMPGRG

which is annotated as comprising a signal peptide (residues 1-19, i.e.,MIHLGHILFLLLLPVAAAQ (SEQ ID NO: 43)) and a mature peptide (residues20-92, i.e TTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQDGKVYINMPGRG (SEQ ID NO: 44)), and is further annotated as comprising atransmembrane region (residues 49-69, i.e., LLAGLVAADAVASLLIVGAVF (SEQID NO: 45)).

As used herein, DAP12 refers to human polypeptide or polynucleotide (ororthologs in other species if the context so indicates) having exemplarysequences as follows. Four human DAP12 transcript variants have beenreported. One transcript variant, human DAP12 isoform 1 precursor (TYROprotein tyrosine kinase-binding protein isoform 1 precursor) has Genbankaccession number NP_003323.1 having the sequence:

(SEQ ID NO: 46) MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQR SDVYSDLNTQRPYYK

which is annotated as comprising a signal peptide (residues 1-27) and amature peptide (residues 28-113), and is further annotated as comprisinga transmembrane region (residues 41-61, i.e., GVLAGIVMGDLVLTVLIALAV (SEQID NO: 47)).

A second transcript variant, human DAP12 isoform 2 precursor (TYROprotein tyrosine kinase-binding protein isoform 2 precursor) has Genbankaccession number NP_937758.1 having the sequence:

(SEQ ID NO: 48) MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESPYQELQGQRS DVYSDLNTQRPYYK

which is annotated as comprising a signal peptide (residues 1-27) and amature peptide (residues 28-112), and is further annotated as comprisinga transmembrane region (residues 41-61, i.e., GVLAGIVMGDLVLTVLIALAV (SEQID NO: 49)).

A third transcript variant, human DAP12 isoform 3 precursor (TYROprotein tyrosine kinase-binding protein isoform 3 precursor) has Genbankaccession number NP_001166985.1 having the sequence:

(SEQ ID NO: 50) MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQR PYYK

which is annotated as comprising a signal peptide (residues 1-24) and amature peptide (residues 25-102).

A fourth transcript variant, human DAP12 isoform 4 precursor (TYROprotein tyrosine kinase-binding protein isoform 4 precursor) has Genbankaccession number NP_001166986.1 having the sequence:

(SEQ ID NO: 51) MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESPYQELQGQRSDVYSDLNTQRP YYK

which is annotated as comprising a signal peptide (residues 1-24) and amature peptide (residues 25-101).

As used herein, CD27 refers to human polypeptide or polynucleotide (ororthologs in other species if the context so indicates) having exemplarysequences as follows. CD27 antigen precursor (also identified in Genbankas “T-cell activation antigen” and “CD27 molecule”) has Genbankaccession numbers NP_001233, AAH12160, and AAA58411, each having thesequence:

(SEQ ID NO: 52) MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCRYSCPREEEGSTI PIQEDYRKPEPACSP

for which exemplary coding sequences include or are contained in GenbankAccession Nos. BC012160.1, M63928.1, and NM_001242.4.

CD27 (NP_001233) is annotated as comprising a signal peptide (residues1-20) and a mature peptide (residues 21-260), and is further annotatedas including a transmembrane region (residues 192-212, i.e.,ILVIFSGMFLVFTLAGALFLH (SEQ ID NO: 53)) and additionally includes thecytoplasmic domain sequence

(SEQ ID NO: 54) QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP.

As used herein, 41BB (also known as tumor necrosis factor receptorsuperfamily member 9, TNFRSF9, 4-1BB, CD137, CDw137, Or ILA) refers tohuman polypeptides or polynucleotides (or orthologs in other species ifthe context so indicates) having exemplary sequences as follows. Tumornecrosis factor receptor superfamily member 9 precursor has Genbankaccession number NP_001552.2, having the sequence:

(SEQ ID NO: 55) MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKCECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCEL

(for which an exemplary coding sequence is contained in NM_001561.5)which polypeptide is annotated as including a signal peptide (residues1-17) and mature peptide (residues 18-255) and is additionally annotatedas including a transmembrane region (residues 187-213, i.e.,HSFFLALTSTALLFLLFFLTLRFSVV (SEQ ID NO: 56)).

As used herein, OX40 (also known as tumor necrosis factor receptorsuperfamily member 4 precursor; ACT35; CD134; TXGP1L) refers to humanpolypeptides or polynucleotides (or orthologs in other species if thecontext so indicates) having exemplary sequences as follows. Tumornecrosis factor receptor superfamily member 4 precursor has Genbankaccession number NP_003318.1, having the sequence:

(SEQ ID NO: 57) MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

(for which an exemplary coding sequence is contained in NM_003327.3)which polypeptide is annotated as including a signal peptide (residues1-28) and mature peptide (residues 29-277) and is additionally annotatedas including a transmembrane region (residues 215-235, i.e.,VAAILGLGLVLGLLGPLAILL (SEQ ID NO: 58)).

It will be appreciated by those of ordinary skill in the art that theendpoints of the above-identified domains can be altered within thescope of the present disclosure, for example by extending or truncatingone or both endpoints, e.g, by up to plus or minus five amino acids andoptionally substituting an omitted portion of a domain with a suitablelinker (such as an engineered or artificial sequence or a functionallysimilar sequence drawn from the same approximate region of a homologousprotein).

In the context of the present disclosure, Chimeric NKp30 refers to apolypeptide (or coding polynucleotide) comprising the ligand-bindingdomain of NKp30 and at least one transmembrane domain and at least onesignaling domain, wherein at least one domain is a domain a polypeptideother than NKp30. Exemplary transmembrane domains comprise thetransmembrane domain of the polypeptides NKp30, CD28, CD8, CD3ζ, DAP10,CD27, and DAP12, preferably CD28, CD8, or CD3ζ, and most preferablyCD28. Exemplary signaling domains comprises the signaling domain (e.g.,cytoplasmic domains) of a polypeptide selected from the groupconsisting: NKp30, CD28, CD8, CD3ζ, DAP10, CD27, and DAP12, such as CD28and/or CD3ζ.

In the context of the present disclosure, a “therapeutically effectiveamount” is identified by one of skill in the art as being an amount ofchimeric NKp30 T cells that, when administered to a patient, alleviatesthe signs and or symptoms of the disease (e.g., cancer, infection, orautoimmune diseases). The actual amount to be administered can bedetermined based on studies done either in vitro or in vivo where thechimeric NKp30 T cells exhibit pharmacological activity against disease.For example, the chimeric NKp30 T cells may inhibit tumor cell growtheither in vitro or in vivo and the amount of chimeric NKp30 T cells thatinhibits such growth is identified as a therapeutically effectiveamount.

In the context of the present disclosure, the words “chimeric,”“chimera” and the phrases “chimeric gene” and “chimeric polypeptide” andthe like refer to a gene comprising an in-frame fusion between codingsequences of different polypeptides or portions of polypeptides, or theprotein produced by translation thereof. Preferred chimeras comprisecomplete domains of different proteins, e.g., comprising one or more ofeach of extracellular, transmembrane, and cytoplasmic domains, whereineach domain is independently selected.

In the context of the present disclosure, the phrase “chimeric NKp30 Tcells” or similar phrases refer to a T cell, which may be an isolated Tcell, which expresses a chimeric NKp30 receptor. Optionally, a chimericNKp30 T cell may also be TCR-deficient T cell, and/or may recombinantlyexpress one or more additional receptors to initiate signaling to Tcells.

The terms “purified,” “substantially purified,” and “isolated” as usedherein refer to the state of being free of other, dissimilar compoundswith which the compound is normally associated in its natural state, sothat the “purified,” “substantially purified,” and “isolated” subjectcomprises at least 0.5%, 1%, 5%, 10%, or 20%, and most preferably atleast 50% or 75% of the mass, by weight, of a given sample. In onepreferred embodiment, these terms refer to the compound comprising atleast 95% of the mass, by weight, of a given sample. As used herein, theterms “purified,” “substantially purified,” and “isolated,” whenreferring to a nucleic acid or protein, also refers to a state ofpurification or concentration different than that which occurs naturallyin the mammalian, especially human body. Any degree of purification orconcentration greater than that which occurs naturally in the mammalian,especially human, body, including (1) the purification from otherassociated structures or compounds or (2) the association withstructures or compounds to which it is not normally associated in themammalian, especially human, body, are within the meaning of “isolated.”The nucleic acid or protein or classes of nucleic acids or proteins,described herein, may be isolated, or otherwise associated withstructures or compounds to which they are not normally associated innature, according to a variety of methods and processes known to thoseof skill in the art.

The term “nucleic acid” or “nucleic acid sequence” refers to adeoxyribonucleotide or ribonucleotide oligonucleotide in either single-or double-stranded form. The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogs of natural nucleotides. Theterm also encompasses nucleic-acid-like structures with syntheticbackbones (see e.g., Oligonucleotides and Analogues, a PracticalApproach, ed. F. Eckstein, Oxford Univ. Press (1991); AntisenseStrategies, Annals of the N.Y. Academy of Sciences, Vol. 600, Eds.Baserga et al. (NYAS 1992); Milligan J. Med. Chem. 36:1923-1937 (1993);Antisense Research and Applications (1993, CRC Press), Mata, Toxicol.Appl. Pharmacol. 144:189-197 (1997); Strauss-Soukup, Biochemistry36:8692-8698 (1997); Samstag, Antisense Nucleic Acid Drug Dev, 6:153-156(1996)) (47-53).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating, e.g., sequences in whichthe third position of one or more selected codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al, Nucleic AcidRes., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)) (54-56).The term nucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “plasma membrane translocation domain” or simply “translocationdomain” means a polypeptide domain that, when incorporated into apolypeptide coding sequence, can with greater efficiency “chaperone”or“translocate” the hybrid (“fusion”) protein to the cell plasmamembrane than without the domain.

The term “signaling domain” refers to a portion of a receptor,costimulatory molecule, or other polypeptide that help effect thefunctional activities of a cell, e.g., a T cell. More specifically, asignaling domain may enhance (or inhibit) the functional activities of aT cell, such as the production of Th1 cytokines, cytotoxicity, in vivopersistence, proliferation, and IFN-g production. Exemplary signalingdomains include the cytoplasmic domains of NKp30, CD28, CD8, CD3ζ,DAP10, CD27, 41BB, OX40, or DAP12, as well as fragments, homologs,variants, analogs, conservatively modified variants, mimetics, and/orchimeras thereof.

The “translocation domain,” “ligand-binding domain,” and chimericreceptors compositions described herein also include “analogs,” or“conservative variants” and “mimetics” (or “peptidomimetics”) withstructures and activity that substantially correspond to the exemplarysequences. Thus, the terms “conservative variant” or “analog” or“mimetic” refer to a polypeptide which has a modified amino acidsequence, such that the change(s) do not substantially alter thepolypeptide's (the conservative variant's) structure and/or activity, asdefined herein. These include conservatively modified variations of anamino acid sequence, i.e., amino acid substitutions, additions ordeletions (such as deletions from one or both ends of a domain, e.g.,deletion of up to five amino acids) of those residues that are notcritical for protein activity, or substitution of amino acids withresidues having similar properties (e.g., acidic, basic, positively ornegatively charged, polar or non-polar, etc.) such that thesubstitutions of even critical amino acids does not substantially alterstructure and/or activity.

More particularly, “conservatively modified variants” applies to bothamino acid and nucleic acid sequences. With respect to particularnucleic acid sequences, conservatively modified variants refers to thosenucleic acids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein, which encodes a polypeptide, also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TOO, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleicacid, which encodes a polypeptide, is implicit in each describedsequence.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. For example, one exemplary guideline toselect conservative substitutions includes (original residue followed byexemplary substitution): ala/gly or ser; arg/lys; asn/gln or his;asp/glu; cys/ser; gin/asn; gly/asp; gly/ala or pro; his/asn or gin;ile/leu or val; leu/ile or val; lys/arg or gln or glu; met/leu or tyr orlie; phe/met or leu or tyr; ser/thr, thr/ser; trp/tyr; tyr/trp or phe;val/ile or leu. An alternative exemplary guideline uses the followingsix groups, each containing amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (I); 5) Isoleucine (T), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); (see also, e.g., Creighton, Proteins, W.H. Freeman andCompany (1984); Schultz and Schimer, Principles of Protein Structure,Springer-Verlag (1979)) (57-58). One of skill in the art will appreciatethat the above-identified substitutions are not the only possibleconservative substitutions. For example, for some purposes, one mayregard all charged amino acids as conservative substitutions for eachother whether they are positive or negative. In addition, individualsubstitutions, deletions or additions that alter, add or delete a singleamino acid or a small percentage of amino acids in an encoded sequencecan also be considered “conservatively modified variations.”

The term “mimetic” and “peptidomimetic” refer to a synthetic chemicalcompound that has substantially the same structural and/or functionalcharacteristics of the polypeptides, e.g., translocation domains,ligand-binding domains, or chimeric receptors. The mimetic can be eitherentirely composed of synthetic, non-natural analogs of amino acids, ormay be chimeric molecules of partly natural peptide amino acids andpartly non-natural analogs of amino acids. The mimetic can alsoincorporate any amount of natural amino acid conservative substitutionsas long as such substitutions also do not substantially alter themimetic's structure and/or activity.

As with polypeptides which are conservative variants, routineexperimentation will determine whether a mimetic's structure and/orfunction is not substantially altered. Polypeptide mimetic compositionscan contain any combination of non-natural structural components, whichare typically from three structural groups: a) residue linkage groupsother than the natural amide bond (“peptide bond”) linkages; b)non-natural residues in place of naturally occurring amino acidresidues; or c) residues which induce secondary structural mimicry,i.e., to induce or stabilize a secondary structure, e.g., a β turn, γturn, β sheet, alpha helix conformation, and the like. A polypeptide canbe characterized as a mimetic when all or some of its residues arejoined by chemical means other than natural peptide bonds. Individualpeptidomimetic residues can be joined by peptide bonds, other chemicalbonds or coupling means, such as, e.g., glutaraldehyde,N-hydroxysuccinimide esters, bifunctional maleimides,N,N′dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(—O)—CH2- and —C(—O)—NH—), aminomethylene (CH2-NH), ethylene, olefin(CH—CH), ether (CH2-O), thioether (CH2-S), tetrazole (CN4), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola, Chemistry andBiochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357,“Peptide Backbone Modifications,” Marcell Dekker, NY (1983)) (157). Apolypeptide can also be characterized as a mimetic by containing all orsome non-natural residues in place of naturally occurring amino acidresidues; non-natural residues are well described in the scientific andpatent literature.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein). Typically, “heterologous nucleic acid” refers to asequence that originates from a source foreign to an intended host cellor, if from the same source, is modified from its original form. Aheterologous nucleic acid in a host cell can comprise a nucleic acidthat is endogenous to the particular host cell but has been modified,for example by mutagenesis or by isolation from native cis-regulatorysequences. A heterologous nucleic acid also includes non-naturallyoccurring multiple copies of a native nucleotide sequence. Aheterologous nucleic acid can also comprise a nucleic acid that isincorporated into a host cell's nucleic acids at a position wherein suchnucleic acids are not ordinarily found.

The term “complementary sequences,” as used herein, indicates twonucleotide sequences that comprise anti-parallel nucleotide sequencescapable of pairing with one another upon formation of hydrogen bondsbetween base pairs. As used herein, the term “complementary sequences”means nucleotide sequences which are substantially complementary, as canbe assessed by the same nucleotide comparison methods set forth below,or is defined as being capable of hybridizing to the nucleic acidsegment in question under relatively stringent conditions such as thosedescribed herein. A particular example of a complementary nucleic acidsegment is an antisense oligonucleotide.

The term “gene” refers broadly to any segment of DNA associated with abiological function. A gene encompasses sequences including but notlimited to a coding sequence, a promoter region, a cis-regulatorysequence, a non-expressed DNA segment that is a specific recognitionsequence for regulatory proteins, a non-expressed DNA segment thatcontributes to gene expression, a DNA segment designed to have desiredparameters, or combinations thereof. A gene can be obtained by a varietyof methods, including cloning from a biological sample, synthesis basedon known or predicted sequence information, and recombinant derivationof an existing sequence.

The term “operatively linked,” as used herein, refers to a functionalcombination between a promoter region and a nucleotide sequence suchthat the transcription of the nucleotide sequence is controlled andregulated by the promoter region. Techniques for operatively linking apromoter region to a nucleotide sequence are known in the art.

The term “vector” is used herein to refer to a nucleic acid moleculehaving nucleotide sequences that enable its replication in a host cell.A vector can also include nucleotide sequences to permit ligation ofnucleotide sequences within the vector, wherein such nucleotidesequences are also replicated in a host cell. Representative vectorsinclude plasmids, cosmids, and viral vectors.

The term “construct,” as used herein to describe a type of constructcomprising an expression construct, refers to a vector furthercomprising a nucleotide sequence operatively inserted with the vector,such that the nucleotide sequence is recombinantly expressed.

The terms “recombinantly expressed” or “recombinantly produced” are usedinterchangeably to refer generally to the process by which a polypeptideencoded by a recombinant nucleic acid is produced.

A “promoter” is defined as an array of nucleic acid sequences thatdirect transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions.

An “inducible” promoter is a promoter that is active under environmentalor developmental regulation. The term “operably linked” refers to afunctional linkage between a nucleic acid expression control sequence(such as a promoter, or array of transcription factor binding sites) anda second nucleic acid sequence, wherein the expression control sequencedirects transcription of the nucleic acid corresponding to the secondsequence.

As used herein, “recombinant” refers to a polynucleotide synthesized orotherwise manipulated in vitro (e.g., “recombinant polynucleotide”), tomethods of using recombinant polynucleotides to produce gene products incells or other biological systems, or to a polypeptide (“recombinantprotein”) encoded by a recombinant polynucleotide. “Recombinant” alsoencompass the ligation of nucleic acids having various coding regions ordomains or promoter sequences from different sources into an expressioncassette or vector for expression of, e.g., inducible or constitutiveexpression of a fusion protein comprising a translocation domain and anucleic acid sequence amplified using a primer.

The term “autoimmunity” or “autoimmune disease or condition” herein An“autoimmune disease” herein is a disease or disorder arising from anddirected against an individual's own tissues or a co-segregate ormanifestation thereof or resulting condition therefrom. Examples ofautoimmune diseases or disorders include, but are not limited toarthritis (rheumatoid arthritis such as acute arthritis, chronicrheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronicinflammatory arthritis, degenerative arthritis, infectious arthritis,Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebralarthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis,arthritis chronica progrediente, arthritis deformans, polyarthritischronica primaria, reactive arthritis, and ankylosing spondylitis),inflammatory hyperproliferative skin diseases, psoriasis such as plaquepsoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of thenails, dermatitis including contact dermatitis, chronic contactdermatitis, allergic dermatitis, allergic contact dermatitis, dermatitisherpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome,urticaria such as chronic allergic urticaria and chronic idiopathicurticaria, including chronic autoimmune urticaria,polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermalnecrolysis, scleroderma (including systemic scleroderma), sclerosis suchas systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS,primary progressive MS (PPMS), and relapsing remitting MS (RRMS),progressive systemic sclerosis, atherosclerosis, arteriosclerosis,sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease(IBD) (for example, Crohn's disease, autoimmune-mediatedgastrointestinal diseases, colitis such as ulcerative colitis, colitisulcerosa, microscopic colitis, collagenous colitis, colitis polyposa,necrotizing enterocolitis, and transmural colitis, and autoimmuneinflammatory bowel disease), pyoderma gangrenosum, erythema nodosum,primary sclerosing cholangitis, episcleritis), respiratory distresssyndrome, including adult or acute respiratory distrss syndrome (ARDS),meningitis, inflammation of all or part of the uvea, iritis,choroiditis, an autoimmune hematological disorder, rheumatoidspondylitis, sudden hearing loss, IgE-mediated diseases such asanaphylaxis and allergic and atopic rhinitis, encephalitis such asRasmussen's encephalitis and limbic and/or brainstem encephalitis,uveitis, such as anterior uveitis, acute anterior uveitis, granulomatousuveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterioruveitis, or autoimmune uveitis, glomerulonephritis (GN) with and withoutnephrotic syndrome such as chronic or acute glomerulonephritis such asprimary GN, immune-mediated GN, membranous GN (membranous nephropathy),idiopathic membranous GN or idiopathic membranous nephropathy, membrano-or membranous proliferative GN (MPGN), including Type I and Type I, andrapidly progressive GN, allergic conditions, allergic reaction, eczemaincluding allergic or atopic eczema, asthma such as asthma bronchiale,bronchial asthma, and auto-immune asthma, conditions involvinginfiltration of T cells and chronic inflammatory responses, chronicpulmonary inflammatory disease, autoimmune myocarditis, leukocyteadhesion deficiency, systemic lupus erythematosus (SLE) or systemiclupus erythematodes such as cutaneous SLE, subacute cutaneous lupuserythematosus, neonatal lupus syndrome (NLE), lupus erythematosusdisseminatus, lupus (including nephritis, cerebritis, pediatric,non-renal, extra-renal, discoid, alopecia), juvenile onset (Type I)diabetes mellitus, including pediatric insulin-dependent diabetesmellitus (IDDM), adult onset diabetes mellitus (Type I diabetes),autoimmune diabetes, idiopathic diabetes insipidus, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis includinglymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis,vasculitides, including vasculitis (including large vessel vasculitis(including polymyalgia rheumatica and giant cell (Takayasu's)arteritis), medium vessel vasculitis (including Kawasaki's disease andpolyarteritis nodosa), microscopic polyarteritis, CNS vasculitis,necrotizing, cutaneous, or hypersensitivity vasculitis, systemicnecrotizing vasculitis, and ANCA-associated vasculitis, such asChurg-Strauss vasculitis or syndrome (CSS)), temporal arteritis,aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia,Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemiaincluding autoimmune hemolytic anemia (AIHA), pernicious anemia (anemiaperniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA),Factor VIII deficiency, hemophilia A, autoimmune neutropenia,pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNSinflammatory disorders, multiple organ injury syndrome such as thosesecondary to septicemia, trauma or hemorrhage, antigen-antibodycomplex-mediated diseases, anti-glomerular basement membrane disease,anti-phospholipid antibody syndrome, allergic neuritis, Bechet's orBehcet's disease, Castleman's syndrome, Goodpasture's syndrome,Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome,pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus(including pemphigus vulgaris, pemphigus foliaceus, pemphigusmucus-membrane pemphigoid, and pemphigus erythematosus), autoimmunepolyendocrinopathies, Reiter's disease or syndrome, immune complexnephritis, antibody-mediated nephritis, neuromyelitis optica,polyneuropathies, chronic neuropathy such as IgM polyneuropathies orIgM-mediated neuropathy, thrombocytopenia (as developed by myocardialinfarction patients, for example), including thrombotic thrombocytopenicpurpura (TTP) and autoimmune or immune-mediated thrombocytopenia such asidiopathic thrombocytopenic purpura (ITP) including chronic or acuteITP, autoimmune disease of the testis and ovary including autoimmuneorchitis and oophoritis, primary hypothyroidism, hypoparathyroidism,autoimmune endocrine diseases including thyroiditis such as autoimmunethyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto'sthyroiditis); or subacute thyroiditis, autoimmune thyroid disease,idiopathic hypothyroidism, Grave's disease, polyglandular syndromes suchas autoimmune polyglandular syndromes (or polyglandular endocrinopathysyndromes), paraneoplastic syndromes, including neurologicparaneoplastic syndromes such as Lambert-Eaton myasthenic syndrome orEaton-Lambert syndrome, stiff-man or stiff-person syndrome,encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergica and experimental allergic encephalomyelitis(EAE), myasthenia gravis such as thymoma-associated myasthenia gravis,cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonusmyoclonus syndrome (OMS), and sensory neuropathy, multifocal motorneuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis,lupoid hepatitis, giant cell hepatitis, chronic active hepatitis orautoimmune chronic active hepatitis, lymphoid interstitial pneumonitis,bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barresyndrome, Berger's disease (IgA nephropathy), idiopathic IgAnephropathy, linear IgA dermatosis, primary biliary cirrhosis,pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease,Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue,idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS;Lou Gehrig's disease), coronary artery disease, autoimmune ear diseasesuch as autoimmune inner ear disease (AGED), autoimmune hearing loss,opsoclonus myoclonus syndrome (OMS), polychondritis such as refractoryor relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis,scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, whichincludes monoclonal B cell lymphocytosis (e.g., benign monoclonalgammopathy and monoclonal garnmopathy of undetermined significance,MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathiessuch as epilepsy, migraine, arrhythmia, muscular disorders, deafness,blindness, periodic paralysis, and channelopathies of the CNS, autism,inflammatory myopathy, focal segmental glomerulosclerosis (FSGS),endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmunehepatological disorder, fibromyalgia, multiple endocrine failure,Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia,demyelinating diseases such as autoimmune demyelinating diseases,diabetic nephropathy, Dressler's syndrome, alopecia greata, CRESTsyndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyl), and telangiectasia), male and female autoimmuneinfertility, mixed connective tissue disease, Chagas' disease, rheumaticfever, recurrent abortion, farmer's lung, erythema multiforme,post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung,allergic granulomatous angiitis, benign lymphocytic angiitis, Alport'ssyndrome, alveolitis such as allergic alveolitis and fibrosingalveolitis, interstitial lung disease, transfusion reaction, leprosy,malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis,aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue,endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonaryfibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis,cystic fibrosis, endophthalmitis, erythema elevatum et diutinum,erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome,Pelty's syndrome, flariasis, cyclitis such as chronic cyclitis,heterochronic cyclitis, iridocyclitis, or Fuch's cyclitis,Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection,echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirusinfection, rubella virus infection, post-vaccination syndromes,congenital rubella infection, Epstein-Barr virus infection, mumps,Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea,post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis,tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrineophthamopathy, chronic hypersensitivity pneumonitis,keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathicnephritic syndrome, minimal change nephropathy, benign familial andischemia-reperfusion injury, retinal autoimmunity, joint inflammation,bronchitis, chronic obstructive airway disease, silicosis, aphthae,aphthous stomatitis, arteriosclerotic disorders, aspermiogenese,autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren'scontracture, endophthalmia phacoanaphylactica, enteritis allergica,erythema nodosum leprosum, idiopathic facial paralysis, chronic fatiguesyndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearingloss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,leucopenia, mononucleosis infectiosa, traverse myelitis, primaryidiopathic myxedema, nephrosis, ophthalmia symphatica, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,infertility due to antispermatozoan antobodies, non-malignant thymoma,vitiligo, SCID and Epstein-Barr virus-associated diseases, acquiredimmune deficiency syndrome (AIDS), parasitic diseases such asLesihmania, toxic-shock syndrome, food poisoning, conditions involvinginfiltration of T cells, leukocyte-adhesion deficiency, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, diseases involving leukocyte diapedesis, multipleorgan injury syndrome, antigen-antibody complex-mediated diseases,antiglomerular basement membrane disease, allergic neuritis, autoimmunepolyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophicgastritis, sympathetic ophthalmia, rheumatic diseases, mixed connectivetissue disease, nephrotic syndrome, insulitis, polyendocrine failure,peripheral neuropathy, autoimmune polyglandular syndrome type I,adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis,dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA),hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosingcholangitis, purulent or nonpurulent sinusitis, acute or chronicsinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, aneosinophil-related disorder such as eosinophilia, pulmonary infiltrationeosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chroniceosinophilic pneumonia, tropical pulmonary cosinophilia,bronchopneumonic aspergillosis, aspergilloma, or granulomas containingeosinophils, anaphylaxis, seronegative spondyloarthritides,polyendocrine autoimmune disease, sclerosing cholangitis, sclera,episclera, chronic mucocutaneous candidiasis, Bruton's syndrome,transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome,ataxia telangiectasia, autoimmune disorders associated with collagendisease, rheumatism, neurological disease, ischemic re-perfusiondisorder, reduction in blood pressure response, vascular dysfunction,antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia,cerebral ischemia, and disease accompanying vascularization, allergichypersensitivity disorders, glomerulonephritides, reperfusion injury,reperfusion injury of myocardial or other tissues, dermatoses with acuteinflammatory components, acute purulent meningitis or other centralnervous system inflammatory disorders, ocular and orbital inflammatorydisorders, granulocyte transfusion-associated syndromes,cytokine-induced toxicity, acute serious inflammation, chronicintractable inflammation, pyelitis, pneumonocirrhosis, diabeticretinopathy, diabetic large-artery disorder, endarterial hyperplasia,peptic ulcer, valvulitis, and endometriosis.

The term “about”, as used herein when referring to a measurable valuesuch as a percentage of sequence identity (e.g., when comparingnucleotide and amino acid sequences as described herein below), anucleotide or protein length, an amount of binding, etc. is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1, and still more preferably 1% from the specified amount,as such variations are appropriate to perform a disclosed method orotherwise carry out an embodiment of the present disclosure.

The term “substantially identical”, is used herein to describe a degreeof similarity between nucleotide sequences, and refers to two or moresequences that have at least about least 60%, preferably at least about70%, more preferably at least about 80%, more preferably about 90% to99%, still more preferably about 95% to about 99%, and most preferablyabout 99% nucleotide identify, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. Preferably, thesubstantial identity exists in nucleotide sequences of at least about100 residues, more preferably in nucleotide sequences of at least about150 residues, and most preferably in nucleotide sequences comprising afull length coding sequence. The term “full length” is used herein torefer to a complete open reading frame encoding a polypeptide, asdescribed further herein. Methods of determining percentage identity arewell known in the art. Preferably, percentage identity is determinedusing the Smith-Waterman alignment algorithm.

In one aspect, substantially identical sequences can be polymorphicsequences. The term “polymorphic” refers to the occurrence of two ormore genetically determined alternative sequences or alleles in apopulation. An allelic difference can be as small as one base pair.

In another aspect, substantially identical sequences can comprisemutagenized sequences, including sequences comprising silent mutations.A mutation can comprise one or more residue changes, a deletion ofresidues, or an insertion of additional residues.

Another indication that two nucleotide sequences are substantiallyidentical is that the two molecules hybridize specifically to orhybridize substantially to each other under stringent conditions. In thecontext of nucleic acid hybridization, two nucleic acid sequences beingcompared can be designated a “probe” and a “target.” A “probe” is areference nucleic acid molecule, and a “target” is a test nucleic acidmolecule, often found within a heterogeneous population of nucleic acidmolecules. A “target sequence” is synonymous with a “test sequence.”

The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex nucleic acid mixture (e.g., total cellular DNA or RNA).

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” and “stringenthybridization wash conditions” refer to conditions under which a probewill hybridize to its target subsequence, typically in a complex mixtureof nucleic acids but to no other sequences. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is that inTigssen, Techniques in Biochemistry and Molecular Biology—HybridizationWith Nucleic Probes, “Overview of principles of hybridization and thestrategy of nucleic acid assays.” (1973) Generally, highly stringenthybridization and wash conditions are selected to be about 5-10° C.lower than the thermal melting point (Tm) for the specific sequence at adefined ionic strength pH. The Tm is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium).

Stringent conditions will be those in which the salt concentration isless than about 1.0 M sodium ion, typically about 0.01 to 1.0M sodiumion concentration (or other salts) at pH 7.0 to 8.3 and the temperatureis at least about 30° C. for short probes (e.g., 10 to 50 nucleotides)and at least about 60° C. for long probes (e.g., greater than 50nucleotides). Stringent conditions may also be achieved with theadditional of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, optionally 10 times background hybridization. Exemplarystringent hybridization conditions are: 50% formamide, 5×SSC, and 1%SDS, incubating at 42° C. or 5×SSC, 1% SDS, incubating at 65° C. Thehybridization and wash steps effected in said exemplary stringenthybridization conditions are each effected for at least 1, 2, 5, 10, 15,30, 60, or more minutes. Preferably, the wash and hybridization stepsare each effected for at least 5 minutes, and more preferably, 10minutes, 15 minutes, or more than 15 minutes.

The phrase “hybridizing substantially to” refers to complementaryhybridization between a probe nucleic acid molecule and a target nucleicacid molecule and embraces minor mismatches that can be accommodated byreducing the stringency of the hybridization media to achieve thedesired hybridization.

An example of stringent hybridization conditions for Southern orNorthern Blot analysis of complementary nucleic acids having more thanabout 100 complementary residues is overnight hybridization in 50%formamide with 1 mg of heparin at 42° C. An example of highly stringentwash conditions is 15 minutes in 0.1×SSC at 65° C. An example ofstringent wash conditions is 15 minutes in 0.2×SSC buffer at 65° C. SeeSambrook et al., eds. (1989) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. for adescription of SSC buffer. Often, a high stringency wash is preceded bya low stringency wash to remove background probe signal. An example ofmedium stringency wash conditions for a duplex of more than about 100nucleotides, is 15 minutes in 1×SSC at 45° C. An example of lowstringency wash for a duplex of more than about 100 nucleotides, is 15minutes in 4× to 6×SSC at 40° C. For short probes (e.g., about 10 to 50nucleotides), stringent conditions typically involve salt concentrationsof less than about 1 M Na+ ion, typically about 0.01 to 1 M Na+ ionconcentration (or other salts) at pH 7.0-8.3, and the temperature istypically at least about 30° C. Stringent conditions can also beachieved with the addition of destabilizing agents such as formamide. Ingeneral, a signal to noise ratio of 2-fold (or higher) than thatobserved for an unrelated probe in the particular hybridization assayindicates detection of a specific hybridization.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially related if the polypeptides that theyencode are substantially related. This occurs, for example, when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. Such hybridizations and wash steps can becarried out for, e.g., 1, 2, 5, 10, 15, 30, 60, or more minutes.Preferably, the wash and hybridization steps are each effected for atleast 5 minutes. A positive hybridization is at least twice background.Those of ordinary skill will readily recognize that alternativehybridization and wash conditions can be utilized to provide conditionsof similar stringency.

In the context of the present disclosure, by a “TCR-deficient T cell”,or a similar phrase is intended an isolated T cell(s) that lacksexpression of a functional TCR, is internally capable of inhibiting itsown TCR production, or produces a TCR that is defective in function sothat the T cell cannot mediate effector functions triggered through TCRrecognition, and further wherein progeny of said T cell(s) may also bereasonably expected to be internally capable of inhibiting their own TCRproduction. Internal capability is important in the context of therapywhere TCR turnover timescales (˜hours) are much faster than demonstrableefficacy timescales (days-months), i.e., internal capability is requiredto maintain the desired phenotype during the therapeutic period. Thismay e.g., be accomplished by different means (for example, as describedin WO2011059836), e.g., by engineering a T cell such that it does notexpress any functional TCR on its cell surface, or by engineering the Tcell such that it does not express one or more of the subunits thatcomprise a functional TCR and therefore does not produce a functionalTCR or by engineering a T cell such that it produces very littlefunctional TCR on its surface, or which expresses a substantiallyimpaired TCR, e.g. by engineering the T cell to express mutated ortruncated forms of one or more of the subunits that comprise the TCR,thereby rendering the T cell incapable of expressing a functional TCR orresulting in a cell that expresses a substantially impaired TCR. Thedifferent subunits that comprise a functional TCR are known in the art(see, e.g., WO2011059836). Whether a cell expresses a functional TCR maybe determined using known assay methods such as are known in the art(Id). By a “substantially impaired TCR” it is meant that this TCR willnot substantially elicit an adverse immune reaction in a host, e.g., aGVHD reaction. As further described in WO2011059836, optionally theseTCR-deficient cells may be engineered to comprise other mutations ortransgenes that e.g., mutations or transgines that affect T cell growthor proliferation, result in expression or absence of expression of adesired gene or gene construct. e.g., another receptor or a cytokine orother immunomodulatory or therapeutic polypeptide or a selectable markersuch as a dominant selectable marker gene, e.g., DHFR or neomycintransferase. For example, a TCR-deficient T cell may express a chimericNKp30 of the present disclosure.

A “pharmaceutical composition” refers to a chemical or biologicalcomposition suitable for administration to a mammal. Such compositionsmay be specifically formulated for administration via one or more of anumber of routes, including but not limited to buccal, intraarterial,intracardial, intracerebroventricular, intradermal, intramuscular,intraocular, intraperitoneal, intraspinal, intrathecal, intravenous,oral, parenteral, rectally via an enema or suppository, subcutaneous,subdermal, sublingual, transdermal, and transmucosal. In addition,administration can occur by means of injection, liquid, gel, drops, orother means of administration.

As used herein, a nucleic acid construct or nucleic acid sequence isintended to mean a DNA molecule which can be transformed or introducedinto a T cell and be transcribed and translated to produce a product(e.g., a chimeric receptor or a suicide protein).

Nucleic acids are “operably linked” when placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for asignal sequence is operably linked to DNA for a polypeptide if it isexpressed as a preprotein that participates in the secretion of thepolypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading frame. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites oralternatively via a PCR/recombination method familiar to those skilledin the art (Gateway® Technology; Invitrogen, Carlsbad Calif.). If suchsites do not exist, the synthetic oligonucleotide adapters or linkersare used in accordance with conventional practice.

The present disclosure contemplates compositions and methods forreducing or ameliorating, or preventing or treating, diseases orconditions such as cancer and infectious disease. In a non-limitingembodiment, the compositions are based on the concept of providing anallogeneic source of isolated human T cells, namely chimeric NKp30 Tcells, which can be manufactured in advance of patient need andinexpensively. The ability to create a single therapeutic product at asingle site using processes that are well controlled is attractive interms of both cost and quality considerations. The change from anautologous to an allogeneic source for T cells offers significantadvantages. For example, it has been estimated that a single healthydonor could supply T cells sufficient to treat dozens of patients aftertransduction and expansion.

As is well known to one of skill in the art, various methods are readilyavailable for isolating allogeneic T cells from a subject, for example,using cell surface marker expression or using commercially availablekits (e.g., ISOCELL™ from Pierce, Rockford, Ill.).

While not necessary for most therapeutic usages of the subject chimericNKp30 T cells, in some instances it may be desirable to remove some orall of the donor T cells from the host shortly after they have mediatedtheir anti-tumor effect. This may be facilitated by engineering the Tcells to express additional receptors or markers that facilitate theirremoval and/or identification in the host such as GFP, suicide genes,and the like. This is not expected to compromise efficacy as it haspreviously been shown that donor T cells do not need to remain long inthe host for a long-term anti-tumor effect to be initiated (Zhang, T.,et al. 2007. Cancer Res. 67:11029-11036; Barber, A. et al. 2008. J.Immunol. 180:72-78).

In one embodiment of the present disclosure, nucleic acid constructsintroduced into engineered T cells further contains a suicide gene suchas thymidine kinase (TK) of the HSV virus (herpes virus) type I (Bonini,et al. (1997) Science 276:1719-1724), a Fas-based “artificial suicidegene” (Thomis, et al. (2001) Blood 97:1249-1257), or E. coli cytosinedeaminase gene which are activated by ganciclovir, AP1903, or5-fluorocytosine, respectively. The suicide gene is advantageouslyincluded in the nucleic acid construct of the present disclosure toprovide for the opportunity to ablate the transduced T cells in case oftoxicity and to destroy the chimeric construct once a tumor has beenreduced or eliminated. The use of suicide genes for eliminatingtransformed or transduced cells is well-known in the art. For example,Bonini, et al. ((1997) Science 276:1719-1724) teach that donorlymphocytes transduced with the HSV-TK suicide gene provide antitumoractivity in patients for up to one year and elimination of thetransduced cells is achieved using ganciclovir. Further, Gonzalez, etal. ((2004) J. Gene Med. 6:704-711) describe the targeting ofneuroblastoma with cytotoxic T lymphocyte clones genetically modified toexpress a chimeric scFvFc:£ immunoreceptor specific for an epitope onLl-CAM, wherein the construct further expresses the hygromycin thymidinekinase (HyTK) suicide gene to eliminate the transgenic clones.

It is contemplated that the suicide gene can be expressed from the samepromoter as the chimeric NKp30, or from a different promoter. Generally,however, nucleic acid sequences encoding the suicide protein andchimeric NKp30 reside on the same construct or vector. Expression of thesuicide gene from the same promoter as the chimeric NKp30 can beaccomplished using any well-known internal ribosome entry site (IRES).Suitable IRES sequences which can be used in the nucleic acid constructof the present disclosure include, but are not limited to, IRES fromEMCV, c-myc, FGF-2, poliovirus and HTLV-1. By way of illustration only,a nucleic acid construct for expressing a chimeric receptor can have thefollowing structure: promoter->chimeric receptor->IRES->suicide gene.Alternatively, the suicide gene can be expressed from a differentpromoter than that of the chimeric receptor (e.g., promoter 1->chimericreceptor->promoter 2->suicide gene).

Because of the broad application of T cells for cell therapies, and theimproved nature of the T cells of the invention, the present disclosureencompasses any method or composition wherein T cells aretherapeutically desirable. Such compositions and methods include thosefor reducing or ameliorating, or preventing or treating cancer,infection, autoimmune diseases, or other diseases or conditions that arebased on the use of T cells derived from an allogeneic source thatexpress a chimeric NKp30.

As indicated, further embodiments of the present disclosure embracerecombinant expression of receptors in said chimeric NKp30 T cells, suchas chimeric NKG2D, chimeric Fv domains, NKG2D, or any other receptor toinitiate signals to T cells, thereby creating potent, specific effectorT cells. One of skill in the art can select the appropriate receptor tobe expressed by the T cell based on the disease to be treated. Forexample, receptors that can be expressed by the T cell for treatment ofcancer would include any receptor to a ligand that has been identifiedon cancer cells. Such receptors include, but are not limited to, NKG2A,NKG2C, NKG2F, LLT1, AICL, CD26, NKRP1, NKp44, NKp46, CD244 (2B4),DNAM-1, and NKp80.

In an exemplary embodiment, the modified T cells do not express afunctional T cell receptor (TCR). In this embodiment, the T cells areTCR-deficient in the expression of a functional TCR. These cells canfunction as a platform to allow the expression of other targetingreceptors (such as chimeric NKp30 as described herein) that may beuseful in specific diseases, while retaining the effector functions of Tcells, albeit without a functioning TCR.

In an exemplary embodiment, the chimeric NKp30 T cells may comprise oneor more additional receptors. Such additional receptors include, but notlimited to, chimeric receptors comprising a ligand binding domainobtained from NKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL, CD26, NKRP1,NKp44, NKp46, CD244 (2B4), DNAM-1, and NKp80, or an anti-tumor antibodysuch as anti-Her2neu or anti-EGFR, and a signaling domain obtained fromCD3ζ, DAP10, CD28, 41BB, CD27, and CD40L. In one embodiment of thepresent disclosure, the chimeric receptor may bind B7-H6, BAT3, Her2neu,EGFR, mesothelin, CD38, CD20, CD19, PSA, MUC1, MUC2, MUC3A, MUC3B, MUC4,MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13, MUC15, MUC16, MUC17,MUC19, MUC20, estrogen receptor, progesterone receptor, or RON. Inadditional exemplary embodiments of the present disclosure, the chimericreceptor may bind MIC-A, MIC-B, or one or more members of the ULBP/RAET1family including ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

In the methods of the present disclosure a patient suffering fromcancer, infection, or autoimmune disease is administered atherapeutically effective amount of a composition comprising saidchimeric NKp30 T cells. In another embodiment of the present disclosure,a therapeutically effective amount of a composition comprising saidchimeric NKp30 T cells is administered to prevent, treat, or reducecancer, infection, or autoimmune diseases.

Methods of Producing TCR-Deficient T-Cells

The chimeric NKp30 T cells may further be a TCR-deficient (i.e., stablylacking expression of a functional TCR). Removal of TCR function mayadvantageously provide “universal” cell products, which can be storedfor future therapy of any patient.

As is further described in WO/2011/05936, TCR-deficient T-cells may beproduced using a variety of approaches. T cells internalize, sort, anddegrade the entire T cell receptor as a complex, with a half-life ofabout 10 hours in resting T cells and 3 hours in stimulated T cells (vonEssen, M. et al. 2004. J. Immunol. 173:384-393). Proper functioning ofthe TCR complex requires the proper stoichiometric ratio of the proteinsthat compose the TCR complex. TCR function is though to require twofunctioning TCR C proteins with ITAM motifs. The activation of the TCRupon engagement of its MHC-peptide ligand requires the engagement ofseveral TCRs on the same T cell, which all must signal properly. Thus,if a TCR complex is destabilized with proteins that do not associateproperly or cannot signal optimally, the T cell will not becomeactivated sufficiently to begin a cellular response.

In one embodiment, TCR expression is eliminated using small-hairpin RNAs(shRNAs) that target nucleic acids encoding specific TCRs (e.g., TCR-αand TCR-β) and/or CD3 chains in primary T cells. By blocking expressionof one or more of these proteins, the T cell will no longer produce oneor more of the key components of the TCR complex, thereby destabilizingthe TCR complex and preventing cell surface expression of a functionalTCR. Even though some TCR complexes can be recycled to the cell surface,the shRNA will prevent new production of TCR proteins resulting indegradation and removal of the entire TCR complex, resulting in theproduction of T cell having a stable deficiency in functional TCRexpression.

Expression of shRNAs in primary T cells can be achieved using anyconventional expression system, e.g., a lentiviral expression system.Although lentiviruses are useful for targeting resting primary T cells,not all T cells will express the shRNAs. Some of these T cells may notexpress sufficient amounts of the shRNAs to allow enough inhibition ofTCR expression to alter the functional activity of the T cell. Thus, Tcells that retain moderate to high TCR expression after viraltransduction can be removed, e.g., by cell sorting or separationtechniques, so that the remaining T cells are deficient in cell surfaceTCR or CD3, enabling the expansion of an isolated population of T cellsdeficient in expression of functional TCR or CD3.

In anon-limiting embodiment, exemplary targeting shRNAs have beendesigned for key components of the TCR complex as set forth below (Table1).

TABLE 1 Tar- SEQ Tar- get GC ID get base shRNA Sequence % NO: TCR-β18^(a) AGTGCGAGGAGATTCGGCAGCTTAT 52  70 21^(a) GCGAGGAGATTCGGCAGCTTATTTC52  71 48^(a) CCACCATCCTCTATGAGATCTTGCT 48  72 54^(a)TCCTCTATGAGATCTTGCTAGGGAA 44  73 TCR-α  3^(b) TCTATGGCTTCAACTGGCTAGGGTG52  74 76^(b) CAGGTAGAGGCCTTGTCCACCTAAT 52  75 01^(b)GCAGCAGACACTGCTTCTTACTTCT 48  76 07^(b) GACACTGCTTCTTACTTCTGTGCTA 44  77CD3-ε 89^(c) CCTCTGCCTCTTATCAGTTGGCGTT 52  78 27^(c)GAGCAAAGTGGTTATTATGTCTGCT 40  79 62^(c) AAGCAAACCAGAAGATGCGAACTTT 40  8045  GACCTGTATTCTGGCCTGAATCAGA 48  81 GGCCTCTGCCTCTTATCAGTT 52  82GCCTCTGCCTCTTATCAGTTG 52  83 GCCTCTTATCAGTTGGCGTTT 48  84AGGATCACCTGTCACTGAAGG 52  85 GGATCACCTGTCACTGAAGGA 52  86GAATTGGAGCAAAGTGGTTAT 38  87 GGAGCAAAGTGGTTATTATGT 38  88GCAAACCAGAAGATGCGAACT 48  89 ACCTGTATTCTGGCCTGAATC 48  90GCCTGAATCAGAGACGCATCT 52  91 CTGAAATACTATGGCAACACAATGATAAA 31  92AAACATAGGCAGTGATGAGGATCACCTGT 45  93 ATTGTCATAGTGGACATCTGCATCACTGG 45 94 CTGTATTCTGGCCTGAATCAGAGACGCAT 48  95 CD3-δ^(d) GATACCTATAGAGGAACTTGA38  96 GACAGAGTGTTTGTGAATTGC 43  97 GAACACTGCTCTCAGACATTA 43  98GGACCCACGAGGAATATATAG 48  99 GGTGTAATGGGACAGATATAT 38 100GCAAGTTCATTATCGAATGTG 38 101 GGCTGGCATCATTGTCACTGA 52 102GCTGGCATCATTGTCACTGAT 48 103 GCATCATTGTCACTGATGTCA 43 104GCTTTGGGAGTCTTCTGCTTT 48 105 TGGAACATAGCACGTTTCTCTCTGGCCTG 52 106CTGCTCTCAGACATTACAAGACTGGACCT 48 107 ACCGTGGCTGGCATCATTGTCACTGATGT 52108 TGATGCTCAGTACAGCCACCTTGGAGGAA 52 109 CD3-γ^(e) GGCTATCATTCTTCTTCAAGG 43 110 GCCCAGTCAATCAAAGGAAAC 48 111GGTTAAGGTGTATGACTATCA 38 112 GGTTCGGTACTTCTGACTTGT 48 113GAATGTGTCAGAACTGCATTG 43 114 GCAGCCACCATATCTGGCTTT 52 115GGCTTTCTCTTTGCTGAAATC 43 116 GCTTTCTCTTTGCTGAAATCG 43 117GCCACCTTCAAGGAAACCAGT 52 118 GAAACCAGTTGAGGAGGAATT 43 119GGCTTTCTCTTTGCTGAAATCGTCAGCAT 45 120 AGGATGGAGTTCGCCAGTCGAGAGCTTCA 55121 CCTCAAGGATCGAGAAGATGACCAGTACA 48 122 TACAGCCACCTTCAAGGAAACCAGTTGAG48 123 ^(a)With reference to Accession No. EU030678. ^(b)With referenceto Accession No. AY247834. ^(c)With reference to Accession No.NM_000733. ^(d)With reference to Accession No. NM_000732. ^(e)Withreference to Accession No. NM_000073.

TCR-α, TCR-β, TCR-γ, TCR-δ, CD3-γ, CD3-ζ, CD3-ε, or CD3ζ mRNAs can betargeted separately or together using a variety of targeting shRNAs. TheTCR-β and TCR-α chains are composed of variable and constant portions.Several targeting shRNAs have been designed for the constant portions ofthese TCR/CD3 sequences. One or a combination of shRNAs can be used foreach molecular target to identify the most efficient inhibitor of TCRexpression. Using established protocols, each shRNA construct is clonedinto, e.g., a pLko.1 plasmid, with expression controlled by a promoterroutinely used in the art, e.g., the U6p promoter. The resultingconstruct can be screened and confirmed for accuracy by sequencing. TheshRNA expression plasmid can then be transfected into any suitable hostcell (e.g., 293T), together with a packaging plasmid and an envelopeplasmid for packaging. Primary human peripheral blood mononuclear cells(PBMCs) are isolated from healthy donors and activated with low dosesoluble anti-CD3 and 25 U/ml rhuIL-2 for 48 hours. Although it is notrequired to activate T cells for lentiviral transduction, transductionworks more efficiently and allows the cells to continue to expand inIL-2. The activated cells are washed and transduced, e.g., using a 1hour spin-fection at 30° C., followed by a 7 hour resting period.

In another embodiment, over-expression of a dominant-negative inhibitorprotein is capable of interrupting TCR expression or function. In thisembodiment, a minigene that incorporates part, or all, of apolynucleotide encoding for one of the TCR components (e.g., TCR-α,TCR-β, CD3-γ, CD3-δ, CD3-ε, or CD3ζ) is prepared, but is modified sothat: (1) it lacks key signaling motifs (e.g. an ITAM) required forprotein function; (2) is modified so it does not associate properly withits other natural TCR components; or (3) can associate properly but doesnot bind ligands (e.g. a truncated TCRβ minigene).

These minigenes may also encode a portion of a protein that serves as ameans to identify the over-expressed minigene. For example,polynucleotides encoding a truncated CD19 protein, which contains thebinding site for anti-CD19 mAbs, can be operably linked to the minigeneso that the resulting cell that expresses the minigene will express theencoded protein and can be identified with anti-CD19 mAbs. Thisidentification enables one to determine the extent of minigeneexpression and isolate cells expressing this protein (and thus lack afunctional TCR).

In one embodiment, over-expression of a minigene lacking a signalingmotif(s) lead to a TCR complex that cannot signal properly when the TCRis engaged by its MHC-peptide ligand on an opposing cell. In anon-limiting embodiment, high expression of this minigene (and theencoded polypeptide) outcompetes the natural complete protein when theTCR components associate, resulting in a TCR complex that cannot signal.In another embodiment, the minigene comprises, or alternatively consistsof, a polynucleotide encoding full or partial CD3ζ, CD3-γ, CD3-δ, orCD3-ε polypeptides lacking the ITAM motifs required for signaling. TheCD3ζ protein contains three ITAM motifs in the cytoplasmic portion, andin one embodiment, removal of one or more of these through truncation ormutation inhibits proper TCR signaling in any complexes where thismodified protein is incorporated. The construct may incorporate IM orother signaling motifs, which are known to alter cell signaling andpromote inhibitory signals through the recruitment of phosphatases suchas SHP1 and SHP2.

In another embodiment, over-expression of a minigene is modified so thatthe encoded polypeptide can associate with some, but not all, of itsnatural partners, creating competition with the normal protein for thoseassociating proteins. In another non-limiting hypothesis, high levelexpression of the minigene (and the encoded polypeptide) outcompetes thenatural partner proteins and prevents assembly of a functional TCRcomplex, which requires all components to associate in the proper ratiosand protein-protein interactions. In another embodiment, minigenescomprise, or alternatively consist of, all or part of thepolynucleotides encoding full-length proteins (e.g., TCR-α, TCR-β,CD3-γ, CD3-δ, CD3-ϵ, or CD3ζ), but containing selected deletions in thesequence coding for amino acids in the transmembrane portion of theprotein that are known to be required for assembly with other TCR/CD3proteins.

In a preferred embodiment, selected deletions in the sequence coding foramino acids in the transmembrane portion of the protein known to berequired for assembly with other TCR/CD3 proteins include, but are notlimited to: the arginine residue at position S in the TCR-αtransmembrane region; the lysine residue at position 10 in the TCR-αtransmembrane region; the lysine residue at position 9 in the TCR-βtransmembrane region; the glutamic acid residue in the transmembraneregion of CD3-γ; the aspartic acid residue in the transmembrane regionof CD3-δ-ε; the aspartic acid residue in the transmembrane region ofCD3-ε; and the aspartic acid residue in the transmembrane region ofCD3ζ.

Over-expression of a truncated TCR-α, TCR-β, TCR-γ, or TCR-δ proteinresults in a TCR complex that cannot bind to MHC-peptide ligands, andthus will not function to activate the T cell. In another embodiment,minigenes comprise, or alternatively consist of, polynucleotidesencoding the entire cytoplasmic and transmembrane portions of theseproteins and portions of the extracellular region, but lackspolynucleotides encoding all or part of the first extracellular domain(i.e., the most outer domain containing the ligand binding site). In apreferred embodiment, said minigene polynucleotides do not encode Vα andVβ polypeptides of the TCR-α and TCR-β chains. In one embodiment, theminigene polynucleotides may be operably linked to polynucleotidesencoding a protein epitope tag (e.g. CD19), thereby allowing mAbidentification of cells expressing these genes.

In another embodiment, the chimeric NKp30 T cells may be TCR-deficientand may express an additional functional, non-TCR receptor, includingthose described below under the heading “additional receptors.”

In another embodiment, these minigenes can be expressed using a strongviral promoter, such as the 5′LTR of a retrovirus, or a CMV or SV40promoter. Typically, this promoter is immediately upstream of theminigene and leads to a high expression of the minigene mRNA. In anotherembodiment, the construct encodes a second polynucleotide sequence underthe same promoter (using for example an IRES DNA sequence between) oranother promoter. This second polynucleotide sequence may encode for afunctional non-TCR receptor providing specificity for the T cell, suchas a chimeric NKp30 of the present disclosure. Additional examples ofthis polynucleotide include, but are not limited to, chimeric NK02D,chimeric NKp30, chimeric NKp46, chimeric anti-CD19, or chimericanti-Her2neu. In a further embodiment, promoter-minigenes areconstructed into a retroviral or other suitable expression plasmid andtransfected or transduced directly into T cells using standard methods(Zhang, T. et al., (2006) Cancer Res., 66(11) 5927-5933; Barber, A. etal., (2007) Cancer Res., 67(10):5003-5008).

After viral transduction and expansion using any of the methodsdiscussed previously, any T cells that still express TCR/CD3 are removedusing anti-CD3 mAbs and magnetic beads using Miltenyi selection columnsas previously described (Barber, A. et al., (2007) Cancer Res.,67(10):5003-5008). The T cells are subsequently washed and cultured inIL-2 (50 U/ml) for 3 to 7 days to allow expansion of the effector cellsin a similar manner as for use of the cells in vivo.

The expression of TCR as and CD3 can be evaluated by flow cytometry andquantitative real-time PCR (QRT-PCR). Expression of TCR-α, TCR-β, CD3ε,CD3ζ, and GAPDH (as a control) mRNA can be analyzed by QRT-PCR using anABI7300 real-time PCR instrument and gene-specific TAQMAN® primers usingmethods similar to those used in Sentman, C. L. et al ((2004) J.Immunol. 173:6760-6766). Changes in cell surface expression can bedetermined using antibodies specific for TCR-α, TCR-β, CD3ε, CD8, CD4,CD5, and CD45.

It is possible that a single shRNA species may not sufficiently inhibitTCR expression on the cell surface. In this case, multiple TCR shRNAsmay be used simultaneously to target multiple components of the TCRcomplex. Each component is required for TCR complex assembly at the cellsurface, so a loss of one of these proteins can result in loss of TCRexpression at the cell surface. While some or even all TCR expressionmay remain, it is the receptor function which determines whether thereceptor induces an immune response. The functional deficiency, ratherthan complete cell surface absence, is the critical measure. In general,the lower the TCR expression, the less likely sufficient TCRcross-linking can occur to lead to T cell activation via the TCRcomplex. While particular embodiments embrace the targeting of TCR-α,TCR-β, and CD3-ε, other components of the TCR complex, such as CD3-γ,CD3-δ, or CD3ζ, can also be targeted.

The primary aim of removing the TCR from the cell surface is to preventthe activation of the T cell to incompatible MHC alleles. To determinewhether the reduction in TCR expression with each shRNA or minigeneconstruct is sufficient to alter T cell function, the T cells can betested for: (1) cell survival in vitro; (2) proliferation in thepresence of mitomycin C-treated allogeneic PBMCs; and (3) cytokineproduction in response to allogeneic PBMCs, anti-CD3 mAbs, or anti-TCRmAbs.

To test cell survival, transduced T cells are propagated in completeRPMI medium with rhuIL-2 (25-50 U/ml). Cells are plated at similardensities at the start of culture and a sample may be removed for cellcounting and viability daily for 7 or more days. To determine whetherthe T cells express sufficient TCR to induce a response againstallogeneic cells, transduced or control T cells are cultured withmitomycin C-treated allogeneic or syngeneic PBMCs. The T cells arepreloaded with CFSE, which is a cell permeable dye that divides equallybetween daughter cells after division. The extent of cell division canbe readily determined by flow cytometry. Another hallmark of T cellactivation is production of cytokines. To determine whether each shRNAconstruct inhibits T cell function, transduced T cells are cultured withanti-CD3 mAbs (2 ng/ml to 5000 ng/ml). After 24 hours, cell-freesupernatants are collected and the amount of TL-2 and IFN-γ produced isquantified by ELISA. PMA/ionomycin are used as a positive control tostimulate the T cells and T cells alone are used as a negative control.

It is possible that removal of TCR-α or TCR-β components may allow thepreferential expansion of TCR-γ/δT cells. These T cells are quite rarein the blood, however the presence of these cells can be determined withanti-TCR-γ/δ antibodies. If there is an outgrowth of these cells, thetargeting of CD3-ε, which is required for cell surface expression ofboth TCR-α/β and TCR-γ/δ at the cell surface, can be used. Both IL-2 andIFN-γ are key effector cytokines that drive T cell expansion andmacrophage activation. Therefore, reduced production of these cytokinesis a sign of a functional defect. It is also possible to measure changesin other cytokines, such as TNF-α. Any reduction in T cell survival uponelimination of TCR expression can be determined by culturing the T cellswith PBMCs, which better reflects the in vivo environment and providessupport for T cell survival.

Additional Receptors

Further embodiments embrace recombinant expression of additionalreceptors in said chimeric NKp30 T cells. Exemplary additional receptorsinclude chNKG2D, chimeric Fv domains, NKG2D, or any other receptor toinitiate signals to T cells, thereby creating potent, specific effectorT cells. One of skill in the art can select the appropriate receptor tobe expressed by the chimeric NKp30 T cells based on the disease to betreated. For example, receptors that can be expressed by the chimericNKp30 T cells for treatment of cancer would include any receptor to aligand that has been identified on cancer cells. Such receptors include,but are not limited to, NKG2D (GENBANK accession number BC039836), NKG2A(GENBANK accession number AF461812), NKG2C (GENBANK accession numberAJ001684), NKG2F, LLT1, AICL, CD26, NKRP1, NKp30 (e.g., GENBANKaccession number AB055881), NKp44 (e.g., GENBANK accession numberAJ225109), NKp46 (e.g., GENBANK accession number AJ001383), CD244 (2B4),DNAM-1, and NKp80.

In another embodiment, such additional receptors include, but notlimited to, chimeric receptors comprising a ligand binding domainobtained from NKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL, CD26, NKRP1,NKp30, NKp44, NKp46, CD244 (2B4), DNAM-1, and NKp80, or an anti-tumorantibody, such as anti-Her2neu and anti-EGFR, and a signaling domainobtained from CD3ζ (e.g., GENBANK accession number NM_198053), DAP10(e.g., GENBANK accession number AF072845), CD28, 41BB, CD27, and/orCD40L.

In a further embodiment, the additional chimeric receptor binds MIC-A,MIC-B, Her2neu, EGFR, mesothelin, CD38, CD20, CD19, PSA, MUC1, MUC2,MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13,MUC15, MUC16, MUC17, MUC19, MUC20, estrogen receptor, progesteronereceptor, RON, or one or more members of the ULBP/RAET1 family includingULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

By way of illustration only, a chimeric NKp30 may be co-expressed withan additional receptor via one or more viral vectors. To achieveco-expression in one vector, expression of the chimeric NKp30 T cellsand additional receptor may be driven by identical or differentpromoters. In another embodiment, if an IRES sequence is used toseparate the genetic elements then only one promoter is used.

A C-type lectin-like NK cell receptor protein particularly suitable foruse in exemplary additional chimeric receptors includes a receptorexpressed on the surface of natural killer cells, wherein upon bindingto its cognate ligand(s) it alters NK cell activation. The receptor maywork alone or in concert with other molecules. Ligands for thesereceptors are generally expressed on the surface of one or more tumorcell types, e.g., tumors associated with cancers of the colon, lung,breast, kidney, ovary, cervix, and prostate; melanomas; myelomas;leukemias; and lymphomas (Wu, et al. (2004) J. Clin. Invest. 114:60-568;Groh, et al. (1999) Proc. Natl. Acad. Sci. USA 96:6879-6884; Pende, etal. (2001) Eur. J. Immunol. 31:1076-1086) and are not widely expressedon the surface of cells of normal tissues. Examples of such ligandsinclude, but are not limited to, MIC-A, MIC-B, heat shock proteins, ULBPbinding proteins (e.g., ULPBs 1-4), and non-classical HLA molecules suchas HLA-E and HLA-G, whereas classical MHC molecules such as HLA-A,HLA-B, or HLA-C and alleles thereof are not generally considered strongligands of the C-type lectin-like NK cell receptor protein. C-typelectin-like NK cell receptors which bind these ligands generally have atype II protein structure, wherein the N-terminal end of the protein iscytoplasmic. In addition to any NK cell receptors previously listedabove, further exemplary NK cell receptors of this type include, but arenot limited to, Dectin-1 (GENBANK accession number AJ312373 orAJ312372), Mast cell function-associated antigen (GENBANK accessionnumber AF097358), HNKR-P1A (GENBANK accession number U11276), LLT1(GENBANK accession number AF133299), CD69 (GENBANK accession numberNM_001781), CD69 homolog, CD72 (GENBANK accession number NM_001782),CD94 (GENBANK accession number NM_002262 or NM_007334), KLRF1 (GENBANKaccession number NM_016523), Oxidised LDL receptor (GENBANK accessionnumber NM_002543), CLEC-1, CLEC-2 (GENBANK accession number NM_016509),NKG2D (GENBANK accession number BC039836), NKG2C (GENBANK accessionnumber AJ001684), NKG2A (GENBANK accession number AF461812), NKG2E(GENBANK accession number AF461157), WUGSC:H_DJ0701016.2, or MyeloidDAP12-associating lectin (MDL-1; GENBANK accession number AJ271684). Inan exemplary embodiment, the NK cell receptor is human NKG2D or humanNKG2C.

Similar type I receptors which would be useful in the chimeric receptorinclude NKp46 (GENBANK accession number AJ001383), NKp30 (includingGENBANK accession number AB055881), or NKp44 (GENBANK accession numberAJ225109).

As an alternative to the C-type lectin-like NK cell receptor protein, aprotein associated with a C-type lectin-like NK cell receptor proteincan be used in the additional chimeric receptor protein. In general,proteins associated with C-type lectin-like NK cell receptor am definedas proteins that interact with the receptor and transduce signalstherefrom. Suitable human proteins which function in this manner furtherinclude, but are not limited to, DAP10 (e.g., GENBANK accession numberAF072845), DAP12 (e.g., GENBANK accession number AF019562) and FcRγ.

To the N-terminus of the C-type lectin-like NK cell receptor may befused an immune signaling receptor having an immunoreceptortyrosine-based activation motif (ITAM),(Asp/Glu)-Xaa-Xaa-Tyr*-Xaa-Xaa-(Ile/Leu)-Xaa₆₋₈-Tyr*-Xaa-Xaa-(Ile/Leu)which is involved in the activation of cellular responses via immunereceptors. Similarly, when employing a protein associated with a C-typelectin-like NK cell receptor, an immune signaling receptor can be fusedto the C-terminus of said protein. Suitable immune signaling receptorsfor use in the chimeric receptor include, but are not limited to, the ζchain of the T-cell receptor, the eta chain which differs from the ζchain only in its most C-terminal exon as a result of alternativesplicing of the ζ mRNA, the δ, γ and ε chains of the T-cell receptor(CD3 chains) and the γ subunit of the FcR1 receptor. In particularembodiments, in addition to immune signaling receptors identifiedpreviously, the immune signaling receptor is CD3ζ (e.g., GENBANKaccession number NM_198053), or human Fcε receptor-γ chain (e.g.,GENBANK accession number M33195) or the cytoplasmic domain or a splicingvariant thereof.

In particular embodiments, said additional receptor may be a chimericreceptor fusion between NKG2D and CD3ζ, or DAP10 and CD3ζ.

As will be appreciated by one of skill in the art, in some instances, afew amino acids at the ends of the C-type lectin-like natural killercell receptor (or protein associated therewith) or immune signalingreceptor can be deleted, usually not more than 10, more usually not morethan 5 residues. Also, it may be desirable to introduce a small numberof amino acids at the borders, usually not more than 10, more usuallynot more than 5 residues. The deletion or insertion of amino acids willusually be as a result of the needs of the construction, providing forconvenient restriction sites, ease of manipulation, improvement inlevels of expression, or the like. In addition, the substitution of oneor more amino acids with a different amino acid can occur for similarreasons, usually not substituting more than about five amino acids inany one domain.

Additional Exemplary Chimeric NKp30 Receptors

By way of illustration, human chimeric receptor molecules composed of anNKp30 extracellular domain in combination with transmembrane and/orcytoplasmic domains of CD3ζ and/or CD28 were generated and expressed inhuman T-cells. Specifically, a gene encoding a chimeric receptorcomprising the extracellular domain of human NKp30 and transmembrane andcytoplasmic domains of human CD3ζ (NKp30-CD3ζ receptor) was generated. Agene encoding a chimeric receptor comprising the extracellular domain ofhuman NKp30, the transmembrane domain of CD28, and cytoplasmic domain ofhuman CD3ζ (NKp30-CD28-CD3ζ receptor) was also generated.

The CD3ζ chain is type I protein with the C-terminus in the cytoplasm(Weissman, et al. (1988) Proc. Natl. Acad. Sci. USA 85:9709-9713). Togenerate a chimeric NKG2D-CD3ζ fusion protein, an initiation codon ATGmay be placed ahead of the coding sequence for the cytoplasmic region ofthe CD3ζ chain (without a stop codon TAA). Upon expression, theorientation of the CD3ζ portion is reversed inside the cells.

Expression Constructs

In the nucleic acid construct encoding the chimeric NKp30 receptor ofthe present disclosure, typically a promoter is operably linked to thenucleic acid sequence encoding the chimeric receptor, i.e., they arepositioned so as to promote transcription of the messenger RNA from theDNA encoding the chimeric receptor. The promoter can be of genomicorigin or synthetically generated. A variety of promoters for use in Tcells are well-known in the art (e.g., the CD4 promoter disclosed byMarodon, et al. (2003) Blood 101(9):3416-23). The promoter can beconstitutive or inducible, where induction is associated with thespecific cell type or a specific level of maturation. Alternatively, anumber of well-known viral promoters are also suitable. Additionalexemplary promoters include the β-actin promoter, SV40 early and latepromoters, immunoglobulin promoter, human cytomegalovirus promoter,retrovirus promoter, and the Friend spleen focus-forming virus promoter.The promoters may or may not be associated with enhancers, wherein theenhancers may be naturally associated with the particular promoter orassociated with a different promoter.

The sequence of the open reading frame encoding the chimeric NKp30receptor of the present disclosure can be obtained from a genomic DNAsource, a cDNA source, or can be synthesized (e.g., via PCR), orcombinations thereof. Depending upon the size of the genomic DNA and thenumber of introns, it may be desirable to use cDNA or a combinationthereof as it is found that introns may stabilize the mRNA or provide Tcell-specific expression (Barthel and Goldfeld (2003) J. Immunol.171(7):3612-9). Also, it may be further advantageous to use endogenousor exogenous non-coding regions to stabilize the mRNA.

For expression of the chimeric NKp30 receptor of the present disclosure,the naturally occurring or endogenous transcriptional initiation regionof the nucleic acid sequence encoding N-terminal component of thechimeric receptor can be used to generate the chimeric receptor in thetarget host. Alternatively, an exogenous transcriptional initiationregion can be used which allows for constitutive or inducibleexpression, wherein expression can be controlled depending upon thetarget host, the level of expression desired, the nature of the targethost, and the like.

Likewise, the signal sequence directing the chimeric NKp30 receptor tothe surface membrane can be the endogenous signal sequence of N-terminalcomponent of the chimeric receptor. Optionally, in some instances, itmay be desirable to exchange this sequence for a different signalsequence. However, the signal sequence selected typically should becompatible with the secretory pathway of T cells so that the chimericreceptor is presented on the surface of the T cell.

Similarly, a termination region can be provided by the naturallyoccurring or endogenous transcriptional termination region of thenucleic acid sequence encoding the C-terminal component of the chimericNKp30 receptor. Alternatively, the termination region can be derivedfrom a different source. For the most part, the source of thetermination region is generally not considered to be critical to theexpression of a recombinant protein and a wide variety of terminationregions can be employed without adversely affecting expression.

The chimeric construct, which encodes the chimeric receptor can beprepared in conventional ways. Since, for the most part, naturalsequences are employed, the natural genes can be isolated andmanipulated so as to allow for the proper joining of the variouscomponents. For example, the nucleic acid sequences encoding theproteins of the chimeric receptor can be isolated by employing thepolymerase chain reaction (PCR), using appropriate primers which resultin amplification of the desired portions of the genes. Alternatively,restriction digests of cloned genes can be used to generate the chimericconstruct. In either case, the sequences can be selected to facilitateassembly of the desired chimera, e.g., by provide for restriction siteswhich are blunt-ended, or have complementary overlaps.

The various manipulations for preparing the chimeric construct can becarried out in vitro and in particular embodiments the chimericconstruct is introduced into vectors for cloning and expression in anappropriate host using standard transformation or transfection methods.Thus, after each manipulation, the resulting construct from joining ofthe DNA sequences can be cloned, the vector isolated, and the sequencescreened to insure that the sequence encodes the desired chimericreceptor. The sequence can be screened by restriction analysis,sequencing, or the like.

It is contemplated that the chimeric construct can be introduced into Tcells as naked DNA or in a suitable vector. Methods of stablytransfecting T cells by electroporation using naked DNA are known in theart. See, e.g., U.S. Pat. No. 6,410,319. Naked DNA generally refers tothe DNA encoding a chimeric receptor of the present disclosure containedin a plasmid expression vector in proper orientation for expression.Advantageously, the use of naked DNA can reduce the time required toproduce T cells expressing the chimeric receptor of the presentdisclosure.

Alternatively, a viral vector (e.g., a retroviral vector, adenoviralvector, adeno-associated viral vector, or lentiviral vector) can be usedto introduce the chimeric construct into T cells. Preferred vectors foruse in accordance with the method of the present disclosure arenon-replicating in the subject's T cells. A large number of vectors areknown which are based on viruses, where the copy number of the virusmaintained in the cell is low enough to maintain the viability of thecell. Illustrative vectors include the pFB-neo vectors (STRATAGENE™) aswell as vectors based on HIV, SV40, EBV, HSV or BPV. Once it isestablished that the transfected or transduced T cell is capable ofexpressing the chimeric receptor as a surface membrane protein with thedesired regulation and at a desired level, it can be determined whetherthe chimeric receptor is functional in the host cell to provide for thedesired signal induction (e.g., production of IFN-γ, TNF-β, GM-CSF uponstimulation with the appropriate ligand).

Primary human PBMCs can be isolated from healthy donors and activatedwith low-dose soluble anti-CD3 and rhuIL-2, anti-CD3/anti-CD28 beads andrhuIL-2, or irradiated antigen presenting cells and rhuIL-2. Although itis not required to activate T cells for lentiviral transduction,transduction is more efficient and the cells continue to expand in IL-2.The activated cells may be washed and transduced as described herein,followed by a resting period. The cells may be washed and cultured inIL-2 for 3 to 7 days to allow expansion of the effector cells in asimilar manner as for use of the cells in vivo.

The expression of TCRαβ, CD3, and NKG2D can be evaluated by flowcytometry and quantitative real-time PCR (QRT-PCR). The number of CD4+and CD8+ T cells can also be determined. Overall cell numbers and thepercentage of chimeric NKp30 T cells can be determined by flowcytometry. Vector-only transduced cells can also be included ascontrols.

After viral transduction and expansion, the chimeric NKp30 T cells canbe separated by mAbs with magnetic beads over Miltenyi columns andidentified and isolated. For example, chimeric NKp30 T cells expressioncan be verified by flow cytometry using anti-NKp30 mAbs or QRT-PCR usingspecific primers for the chimeric NKp30 T cells receptor. Function ofthese chimeric NKp30 T cells can be determined by culturing the cellswith tumor cells that express NKp30 ligand(s). T cell proliferation andcytokine production (IFN-γ and IL-2) can be determined by flow cytometryand ELISA, respectively.

Another hallmark of T cell activation is production of cytokines. Todetermine whether chimeric NKp30 T cells induce T cell activation, the Tcells may be co-cultured with syngeneic PBMCs or with tumor cells thatdo (or do not) express NKp30 ligand(s). After a period of incubation,such as 24 hours, cell-free supernatants may be collected and the amountof IL-2 and IFN-γ produced is quantified by ELISA. T cells alone andculture with ligand-deficient cells can be used as a negative control.

Subsequently, the transduced T cells may be reintroduced or administeredto the subject to activate anti-tumor responses in said subject. Tofacilitate administration, the transduced T cells according to thepresent disclosure can be made into a pharmaceutical composition or madeimplant appropriate for administration in vivo, with appropriatecarriers or diluents, which further can be pharmaceutically acceptable.The means of making such a composition or an implant have been describedin the art (see, for instance, Remington's Pharmaceutical Sciences, 16thEd., Mack, ed. (1980)). Where appropriate, the transduced T cells can beformulated into a preparation in semisolid or liquid form, such as acapsule, solution, injection, inhalant, or aerosol, in the usual waysfor their respective route of administration. Means known in the art canbe utilized to prevent or minimize release and absorption of thecomposition until it reaches the target tissue or organ, or to ensuretimed-release of the composition. Desirably, however, a pharmaceuticallyacceptable form is employed which does not render ineffectual the cellsexpressing the chimeric receptor. Thus, desirably the transduced T cellscan be made into a pharmaceutical composition containing a balanced saltsolution, preferably Hanks' balanced salt solution, or normal saline.

Methods of Ameliorating or Reducing Symptoms of, or Treating, orPreventing, Diseases and Disorders Using chimeric NKp30 T cells

The present disclosure is also directed to methods of reducing orameliorating, or preventing or treating, diseases and disorders usingthe chimeric NKp30 T cells described herein, isolated populationsthereof, or therapeutic compositions comprising the same. In oneembodiment, the chimeric NKp30 T cells described herein, isolatedpopulations thereof, or therapeutic compositions comprising the same areused to reduce or ameliorate, or prevent or treat cancer, infection, orautoimmune disease. Additional exemplary diseases and disorders that maypotentially be reduced, ameliorated, prevented, and/or treated using themethods and compositions of the present disclosure can be identified bythose of skill in the art and may include (by way of non-limitingexamples) graft versus host disease (GVHD), transplantation rejection,one or more autoimmune disorders, or radiation sickness. For example,because NKp30 ligands can be expressed by human dendritic cells, it isfurther contemplated that the methods and compositions of the presentdisclosure may be useful to prevent or treat diseases where eliminationof dendritic cells may be helpful, such as autoimmune diseases orrejection of transplanted organs.

Preferably, the cancer expresses at least one NKp30 ligand (e.g.,B7-H6), and more preferably expresses a sufficiently high level of atleast one NKp30 ligand to activate the chimeric NKp30 T cells of thepresent disclosure. Though some cancers may not express an NKp30 ligand,expression of NKp30 ligands have been reported in cell lines from manycancers, including leukemia, lymphomas, cervical cancer, gastricsarcoma, breast cancer, pancreatic cancer, melanoma, and prostatecancer. Optionally, the methods of the present disclosure may include astep of determining susceptibility of the cancer to a therapy disclosedherein, e.g., by detecting expression of one or more NKp30 ligands oncancer cells.

In addition to the illustrative chimeric NKp30 T cells described herein,it is contemplated that chimeric NKp30 T cells can be modified ordeveloped to express additional functional receptors useful in treatmentof diseases such as cancer, infection, or autoimmune diseases, asdescribed previously. Briefly, the treatment methods of the presentdisclosure contemplate the use of chimeric NKp30 T cells expressingadditional receptors, such as chNKG2D, chimeric Fv domains, NKG2D, orany other receptor to initiate signals to T cells, thereby creatingpotent, specific effector T cells. One of skill in the art can selectthe appropriate receptor to be expressed by T cell based on the diseaseto be treated. For example, receptors that can be expressed by thechimeric NKp30 T cells for treatment of cancer would include anyreceptor that binds to a ligand that has been identified on cancercells. Such receptors include, but are not limited to, NKG2D, NKG2A,NKG2C, NKG2F, LLT1, AICL, CD26, NKRP1, NKp30, NKp44, NKp46, CD244 (2B4),DNAM-1, and NKp80.

In another embodiment, such receptors include, but not limited to,chimeric receptors comprising a ligand binding domain obtained fromNKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL, CD26, NKRP1, NKp30, NKp44,NKp46, CD244 (2B4), DNAM-1, and NKp80, or an anti-tumor antibody such asanti-Her2neu and anti-EGFR, and a signaling domain obtained from CD3ζ,DAP10, CD28, 41BB, CD27, and CD40L.

In a further embodiment, the additional receptor binds MIC-A, MIC-B,Her2neu, EGFR, mesothelin, CD38, CD20, CD19, PSA, MUC1, MUC2, MUC3A,MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13, MUC15,MUC16, MUC17, MUC19, MUC20, estrogen receptor, progesterone receptor,RON, or one or more members of the ULBP/RAET1 family including ULBP1,ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

Efficacy of the compositions of the present disclosure can bedemonstrated in the most appropriate in vivo model system depending onthe type of drug product being developed. The medical literatureprovides detailed disclosure on the advantages and uses of a widevariety of such models. For example, there are many different types ofcancer models that are used routinely to examine the pharmacologicalactivity of drugs against cancer such as xenograft mouse models (e.g.,Mattern, J. et al. 1988. Cancer Metastasis Rev. 7:263-284; Macor, P. etal. 2008. Curr. Pharm. Des. 14:2023-2039) or even the inhibition oftumor cell growth in vitro. In the case of GVHD, there are models inmice of both acute GVHD (e.g., He, S. et al. 2008. J. Immunol.181:7581-7592) and chronic GVHD (e.g., Xiao, Z. Y. et al. 2007. LifeSci. 81:1403-1410).

Once the compositions of the present disclosure have been shown to beeffective in vivo in animals, clinical studies may be designed based onthe doses shown to be safe and effective in animals. One of skill in theart can design such clinical studies using standard protocols asdescribed in textbooks such as Spilker (2000. Guide to Clinical Trials.Lippincott Williams & Wilkins: Philadelphia).

Administration

In one embodiment of the present disclosure, the chimeric NKp30 T cellsare administered to a recipient subject at an amount of between about10⁶ to 10¹¹ cells. In a preferred embodiment of the present disclosure,the chimeric NKp30 T cells are administered to a recipient subject at anamount of between 10⁸ to 10⁹ cells. In a preferred embodiment of thepresent disclosure, the chimeric NKp30 T cells are administered to arecipient subject with a frequency of once every twenty-six weeks orless, such as once every sixteen weeks or less, once every eight weeksor less, or once every four weeks or less.

These values provide general guidance of the range of transduced T cellsto be utilized by the practitioner upon optimizing the method of thepresent disclosure for practice. The recitation herein of such ranges byno means precludes the use of a higher or lower amount of a component,as might be warranted in a particular application. For example, theactual dose and schedule can vary depending on whether the compositionsare administered in combination with other pharmaceutical compositions,or depending on inter-individual differences in pharmacokinetics, drugdisposition, and metabolism. One skilled in the art readily can make anynecessary adjustments in accordance with the exigencies of theparticular situation.

A person of skill in the art would be able to determine an effectivedosage and frequency of administration based on teachings in the art orthrough routine experimentation, for example guided by the disclosureherein and the teachings in Goodman, L. S., Gilman, A., Brunton, L. L.,Lazo, J. S., & Parker, K. L. (2006). Goodman & Gilman's thepharmacological basis of therapeutics. New York: McGraw-Hill; Howland,R. D., Mycek, M. J., Harvey, R. A., Champe, P. C., & Mycek, M. J.(2006). Pharmacology. Lippincott's illustrated reviews. Philadelphia:Lippincott Williams & Wilkins; and Golan, D. E. (2008). Principles ofpharmacology: the pathophysiologic basis of drug therapy. Philadelphia,Pa. [etc.]: Lippincott Williams & Wilkins. The dosing schedule can bebased on well-established cell-based therapies (see, e.g., Topalian andRosenberg (1987) Acta Hacmatol. 78 Suppl 1:75-6; U.S. Pat. No.4,690,915) or an alternate continuous infusion strategy can be employed.

In another embodiment of the present disclosure, the chimeric NKp30 Tcells are administered to a subject in a pharmaceutical formulation.

In one embodiment of the present disclosure, the chimeric NKp30 T cellsmay be optionally administered in combination with one or more activeagents. Such active agents include analgesic, antipyretic,anti-inflammatory, antibiotic, antiviral, and anti-cytokine agents.Active agents include agonists, antagonists, and modulators of TNF-α,IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-α, IFN-γ, BAFF,CXCL13, IP-10, VEGF, EPO, EGF, HRG, Hepatocyte Growth Factor (HGF),Hepcidin, including antibodies reactive against any of the foregoing,and antibodies reactive against any of their receptors. Active agentsalso include 2-Arylpropionic acids, Aceclofenac, Acemetacin,Acetylsalicylic acid (Aspirin), Alclofenac, Alminoprofen, Amoxiprin,Ampyrone, Arylalkanoic acids, Azapropazone, Benorylate/Benorilate,Benoxaprofen, Bromfenac, Carprofen, Celecoxib, Choline magnesiumsalicylate, Clofezone, COX-2 inhibitors, Dexibuprofen, Dexketoprofen,Diclofenac, Diflunisal, Droxicam, Ethenzamide, Etodolac, Etoricoxib,Faislamine, fenamic acids, Fenbufen, Fenoprofen, Flufenamic acid,Flunoxaprofen, Flurbiprofen, Ibuprofen, Ibuproxam, Indometacin,Indoprofen, Kebuzone, Ketoprofen, Ketorolac, Lornoxicam, Loxoprofen,Lumiracoxib, Magnesium salicylate, Meclofenamic acid, Mefenamic acid,Meloxicam, Metamizole, Methyl salicylate, Mofebutazone, Nabumetone,Naproxen, N-Arylanthranilic acids, Nerve Growth Factor (NGF),Oxametacin, Oxaprozin, Oxicams, Oxyphenbutazone, Parecoxib, Phenazone,Phenylbutazone, Phenylbutazone, Piroxicam, Pirprofen, profens,Proglumetacin, Pyrazolidine derivatives, Rofecoxib, Salicyl salicylate,Salicylamide, Salicylates, Sufinpyrazone, Sulindac, Suprofen, Tenoxicam,Tiaprofenic acid, Tolfenamic acid, Tolmetin, and Valdecoxib.

Antibiotics include Amikacin, Aminoglycosides, Amoxicillin, Ampicillin,Ansamycins, Arsphenamine, Azithromycin, Azlocillin, Aztreonam,Bacitracin, Carbacephem, Carbapenems, Carbenicillin, Cefaclor,Cefadroxil, Cefalexin, Cefaothin, Cefalotin, Cefamandole, Cefazolin,Cefdinir, Cefditoren, Cefepime, Cefixime, Cefoperazone, Cefotaxime,Cefoxitin, Cefpodoxime, Cefprozil, Ceftazidime, Ceftibuten, Ceftizoxime,Ceftobiprole, Ceftriaxone, Cefuroxime, Cephalosporins, Chloramphenicol,Cilastatin, Ciprofloxacin, Clarithromycin, Clindamycin, Cloxacillin,Colistin, Co-trimoxazole, Dalfopristin, Demeclocycline, Dicloxacillin,Dirithromycin, Doripenem, Doxycycline, Enoxacin, Ertapenem,Erythromycin, Ethambutol, Flucloxacillin, Fosfomycin, Furazolidone,Fusidic acid, Gatifloxacin, Geldanamycin, Gentamicin, Glycopeptides,Herbimycin, Imipenem, Isoniazid, Kanamycin, Levofloxacin, Lincomycin,Linezolid, Lomefloxacin, Loracarbef, Macrolides, Mafenide, Meropenem,Meticillin, Metronidazole, Mezlocillin, Minocycline, Monobactams,Moxifloxacin, Mupirocin, Nafcillin, Neomycin, Netilmicin,Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Oxytetracycline,Paromomycin, Penicillin, Penicillins, Piperacillin, Platensimycin,Polymyxin B, Polypeptides, Prontosil, Pyrazinamide, Quinolones,Quinupristin, Rifampicin, Rifampin, Roxithromycin, Spectinomycin,Streptomycin, Sulfacetamide, Sulfamethizole, Sulfanilimide,Sulfasalazine, Sulfisoxazole, Sulfonamides, Teicoplanin, Telithromycin,Tetracycline, Tetracyclines, Ticarcillin, Tinidazole, Tobramycin,Trimethoprim, Trimethoprim-Sulfamethoxazole, Troleandomycin,Trovafloxacin, and Vancomycin.

Active agents also include Aldosterone, Beclometasone, Betamethasone,Corticosteroids, Cortisol, Cortisone acetate, Deoxycorticosteroneacetate, Dexamethasone, Fludrocortisone acetate, Glucocorticoids,Hydrocortisone, Methylprednisolone, Prednisolone, Prednisone, Steroids,and Triamcinolone. Any suitable combination of these active agents isalso contemplated.

A “pharmaceutical excipient” or a “pharmaceutically acceptableexcipient” is a carrier, usually a liquid, in which an activetherapeutic agent is formulated. In one embodiment of the presentdisclosure, the active therapeutic agent is a population of chimericNKp30 T cells. In one embodiment of the present disclosure, the activetherapeutic agent is a population of chimeric NKp30 T cells that areTCR-deficient. The excipient generally does not provide anypharmacological activity to the formulation, though it may providechemical and/or biological stability. Exemplary formulations can befound, for example, in Remington's Pharmaceutical Sciences, 19^(th) Ed.,Grennaro, A., Ed., 1995 which is incorporated by reference.

As used herein “pharmaceutically acceptable carrier” or “excipient”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents that arephysiologically compatible. In one embodiment, the carrier is suitablefor parenteral administration. Alternatively, the carrier can besuitable for intravenous, intraperitoneal, intramuscular, or sublingualadministration. Pharmaceutically acceptable carriers include sterileaqueous solutions or dispersions for the extemporaneous preparation ofsterile injectable solutions or dispersions. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe present disclosure is contemplated. Supplementary active compoundscan also be incorporated into the compositions.

In a particularly preferred embodiment of the present disclosure,appropriate carriers include, but are not limited to, Hank's BalancedSalt Solution (HBSS), Phosphate Buffered Saline (PBS), or any freezingmedium having for example, 10% DMSO and 90% human serum.

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage (in this context, the term“sterile” does not preclude presence of intended living components,e.g., live T cells or viruses adapted to transduce a chimeric NKp30receptor of the present disclosure). The composition can be formulatedas a solution. The carrier can be a dispersion medium containing, forexample, water, saline solution, preservatives, etc.

For each of the recited embodiments, the compounds can be administeredby a variety of dosage forms. Any biologically-acceptable dosage formknown to persons of ordinary skill in the art, and combinations thereof,are contemplated. Examples of such dosage forms include, withoutlimitation, liquids, solutions, suspensions, emulsions, injectables(including subcutaneous, intramuscular, intravenous, and intradermal),infusions, and combinations thereof.

The above description of various illustrated embodiments of theinvention is not intended to be exhaustive or to limit the invention tothe precise form disclosed. While specific embodiments of, and examplesfor, the invention are described herein for illustrative purposes,various equivalent modifications are possible within the scope of theinvention, as those skilled in the relevant art will recognize. Theteachings provided herein of the invention can be applied to otherpurposes, other than the examples described above.

These and other changes can be made to the invention in light of theabove detailed description. In general, in the following claims, theterms used should not be construed to limit the invention to thespecific embodiments disclosed in the specification and the claims.Accordingly, the invention is not limited by the disclosure, but insteadthe scope of the invention is to be determined entirely by the followingclaims.

The invention may be practiced in ways other than those particularlydescribed in the foregoing description and examples. Numerousmodifications and variations of the invention are possible in light ofthe above teachings and, therefore, are within the scope of the appendedclaims.

Certain teachings related to T-cell receptor deficient T-cellcompositions and methods of use thereof were disclosed in U.S.Provisional patent application No. 61/255,980, filed Oct. 29, 2009, thedisclosure of which is herein incorporated by reference in its entirety.

Certain teachings related to the production of T cells expressingchimeric receptors and methods of use thereof were disclosed in U.S.patent application publication no. US 2010/0029749, published Feb. 4,2010, the disclosure of which is herein incorporated by reference in itsentirety.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, manuals, books, or otherdisclosures) in the present application, including the Background,Detailed Description, and Examples, are each herein incorporated byreference in their entireties.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

EXAMPLES Example 1: Production of Chimeric NKp30 Human T Cells

Natural killer (NK) cells can attack tumor cells, which are recognizedutilizing a combination of signals from activating and inhibitoryreceptors including NKp30. FIG. 1 provides an graphical overviewproteins involved in NKp30 signaling, which include FcRγ and CD3ζ, eachof which contain immunoreceptor tyrosine-based activation motif (ITAM)which are involved in the activation of cellular responses via immunereceptors.

In an effort to develop a new mechanism for T cells to attack tumorcells and induce host anti-tumor immunity, we genetically modifiedprimary human T cells with chimeric NKp30 receptor containing the(cytoplasmic) CD3ζ and/or CD28 chain signaling domains. Our hypothesiswas that the chimeric NKp30-modified T cells would react to NKp30ligand-positive tumor cells and become fully activated resulting inelimination of the tumor and induction of host anti-tumor immunity. Thechimeric NKp30 constructs used in this study are schematicallyillustrated in IG. 2. Wild-type (WT) NKp30 contains the full-lengthhuman NKp30. The NKp30-CD3ζ receptor comprises the extracellular domainof human NKp30 fused to the transmembrane (TM) and signaling domains ofhuman CD3ζ chain as illustrated in FIG. 16. The NKp30-CD28-CD3ζ receptorwas constructed by joining the extracellular domain of NKp30 to the TMand cytoplasmic domain of human CD28 and further to the signalingdomains of human CD3ζ chain as illustrated in FIG. 17. Due to the natureof dimerization of CD28 molecule, it is thought to be likely thatNKp30-CD28-CD3ζ receptor expresses as a dimer.

Surface expression of chimeric NKp30 receptors on human T cells wasanalyzed by flow cytometry using anti-NKp30 and anti-CD4 mAbs. CD4+ andCD4− populations were clearly discernible in each sample. As expected,mock-transfected cells had little activation (FIG. 3A). Retroviraltransduction of human T cells with wild-type (i.e., non-chimeric) NKp30gene only led to marginal surface expression (FIG. 3B). The chimericconstruct NKp30-CD3ζ (FIG. 3C) was expressed on human T cells atefficiency around 20% (i.e., about 20% of the total cell population hadstaining intensity above a cutoff chosen to indicate positive cellstaining, indicated in each panel by a vertical line). The chimericNKp30-CD28-CD3ζ receptor (FIG. 3D) gave rise to significantly highersurface expression of NKp30 (about 70% of the total cell population).

Example 2: Chimeric NKp30 Human T Cells Specifically Respond to TumorCells Expressing NKp30 Ligands

This example demonstrates that the NKp30 chimeric T cells described inExample 1 were specifically activated by cells expressing NKp30 ligands.

A panel of human tumor cell lines as well as human PBMCs were screenedfor the expression of NKp30 ligands on mRNA level by RT-PCR (FIG. 4)using primers specific for the NKp30 ligands BAT3 and B7-H6, as well asa housekeeping gene (GAPDH) as a positive control. All tested humantumor cells had detectable amounts of BAT3 mRNA, whereas B7-H6expression was readily detected in HeLa, U937, Panc-1, T47D, RPMI8226,K562, and A375 cells, but expression was at lower levels or undetectablein MCF-7, DU145, IM9, U266 and human PBMCs.

Surface expression of NKp30 ligands was then determined using a solublehuman NKp30-mIgG2a fusion protein, which labels cells thatsurface-express NKp30 ligands (such as B7-H6). As shown in FIG. 5, K562,A375 and HeLa cells express high amounts of NKp30 ligands, whereas U937,RPMP8226, T47D and Panc-1 cells express marginal levels of NKp30ligands. Some tumor cells (IM9, MM.1s, MCF-7 and DU145) as well as humanPBMCs do not express NKp30 ligands. B7-H6 mRNA amounts are correlatedwith surface expression of NKp30 ligands, suggesting that B7-H6 is themajor surface ligand of NKp30 in the tested tumors. The result is alsoconsistent with prior reports that BAT3 is a nuclear protein, which isusually not expressed on cell surface and therefore would not beexpected to trigger NKp30 and be detected in this assay.

The murine lymphoma cell line RMA was also negative for NKp30 binding(FIG. 5, “RMA), however, RMA cells transfected with a B7-H6 constructwere positive for NKp30 binding (FIG. 5, “RMA/B7-H6”).

Chimeric NKp30-transduced human T cells were then shown to specificallyrecognize these NKp30 ligand-positive tumor cells and respond byproducing IFN-γ. As shown in FIG. 6. NKp30-CD3ζ+ (grey bars, middle barin each group) or NKp30-CD28-CD3ζ+ (black bars, rightmost bar in eachgroup) T cells produced significant amounts of IFN-γ after co-culturewith NKp30 ligand-positive cells but not with ligand-negative cellsindicating that these chimeric NKp30-modified T cells could functionallyrecognize NKp30 ligand-bearing tumor cells. In contrast, wild-typeNKp30-modified T cells (white bars, left bar in each group) did not showany significant response to the stimulation by NKp30-ligand positivecells. NKp30-CD28-CD3ζ+ evoked significantly higher production of IFN-γin the presence of NKp30 ligand-positive tumor cells than NKp30-CD3ζ,especially for cell lines for which the ligand expression was low. Thereason for this may be higher amount of surface expression and/orpresence of the CD28 co-stimulatory signaling domain ofNKp30-CD28-CD3ζ+.

For the experiments shown in FIG. 6 when tumor cells were grown insuspension, co-culture with gene-modified primary human T cells (10) wasperformed in round-bottom 96-well plates at a ratio of 1:1, whereasadherent tumor cells (2.5×10⁴) were co-cultured with T cells inflat-bottom plates. Tumor cells were irradiated (120 Gys) before use.Cell-free supernatants were collected after 24 hr and analyzed for IFN-γby ELISA using Duoset ELISA kits (R&D systems, Minneapolis, Minn.).

Chimeric NKp30-bearing human T cells were then demonstrated to lyseNKp30-ligand positive tumor cells. The cytotoxic activity of chimericNKp30-modified human T cells against various tumor cell lines wasdetermined using a LDH release assay. NKp30 receptor-modified human Tcells were co-cultured with NKp30 ligand-positive RMA/B7-H6 (murinelymphoma), K562, U937, Hela, Panc-1, A375, T47D cells and -negative RMAat E:T ratios of 5:1 for 5 h. FIG. 7A shows results of triplicateexperiments (mean+/−SD). NKp30-CD3ζ+ (black bars, middle bar in eachgroup) or NKp30-CD28-CD3ζ+ (dark gray bars, rightmost bar in each group)T cells lysed NKp30 ligand-positive cells (RMA/B7-H6, K562, U937, HeLa,Panc-1, A375, and T47D) but not ligand-negative cells (cell line RMA)indicating that these chimeric NKp30-modified T cells could functionallyrecognize NKp30 ligand-bearing tumor cells in a specific manner. Incontrast, a far lower percentage of tumor cells were lysed in thepresence of wild-type NKp30-modified T cells (light gray bars, left barin each group). NKp30-CD28-CD3ζ+ killed a greater percentage of NKp30ligand-positive tumor cells than NKp30-CD3ζ.

Cross-linking of CD28 leads to activation of the PI3K pathway.Therefore, we hypothesized that significantly enhanced IFN-γ productionand cytotoxicity by T cells after engagement of the NKp30-CD28-3ζreceptor might be due to activation of PI3K. To test whether activationof PI3 kinase pathway plays a role in NKp30-CD28-CD3ζ-mediatedcytotoxicity, a PI3K inhibitor LY294002 (10 μM) was pre-incubated with Tcells for 1 hr prior to co-culture with tumor cells expressing B7-H6 atan E:T ratio of 3:1. DMSO (0.02%) was used as vesicle control. Thecytotoxicity was determined by a 5-hr LDH release assay. Results areshown in mean+SD of triplicates (FIG. 8). *: P<0.05. Specific lysis wassignificantly decreased for the chimeric NKp30-CD28-CD3ζ T cellsincubated with the Ly294002 inhibitor, indicating that the PI3 kinaseplays a role in NKp30-CD28-CD3ζ-mediated cytotoxicity. This was notobserved for the chimeric NKp30-CD3ζ T cells. This result was expectedbecause CD28 is thought to induce signals in T cells in a PI3-kinasedependent manner. These results demonstrate that that PI3K is importantfor enhancement of function when the cytoplasmic part of CD28 isincluded. Without intent to be limited by theory, it is believed thatthis construct uses the CD28 induced PI3K signals, such that the PI3Kinhibitor decreases cytotoxicity of the NKp30-CD28-CD3ζ receptor to theamount similar to the NKp30-CD3ζ receptor, which lacks the CD28signaling element. However, irrespective of the mechanism of action, theCD28-containing construct exhibits elevated cytotoxicity (and as shownbelow, tumor protection) relative to other exemplified constructs.

Example 3: Production of Chimeric NKp30 Mouse T Cells

This example demonstrates that human NKp30 receptors (both wild-type andchimeric) can express on primary mouse T cells. Because NKp30 is apseudogene in inbred mice, we determined whether chimeric human NKp30receptors could be expressed on mouse T cells, which allows the testingof in vivo efficacy of chimeric NKp30-modified T cells against NKp30ligand-positive tumor cells in immunocompetent mouse models. One dayafter ConA (1 ug/ml) stimulation, mouse spleen cells were transducedwith retroviral supernatants containing NKp30 or control viruses. Afterexpansion and G418 selection, mouse T cells were stained with anti-CD4FITC and anti-NKp30-PE (FIG. 9). Similar to the expression profileobserved on human T cells, chimeric NKp30 receptors were expressed onmouse T cells, although the expression of NKp30 observed was slightlylower than with human T cells.

As expected, mock-transfected cells had low levels of NKp30 signaling(FIG. 9A). Retroviral transduction of mouse T cells with wild-type(i.e., non-chimeric) NKp30 gene only led to expression on about 12.8% ofcells (FIG. 3B). The chimeric construct NKp30-CD3ζ (FIG. 3C) wasexpressed on mouse T cells at efficiency around 16.7% (i.e., about 16.7%of the total cell population had staining intensity above a cutoffchosen to indicate positive cell staining, indicated in each panel by avertical line). The chimeric NKp30-CD28-CD3ζ receptor (FIG. 9D) gaverise to significantly higher surface expression of NKp30 (about 41.1% ofthe total cell population).

Example 4: Hunan NKp30 Receptors are Functional in Mouse T Cells

This example demonstrates that chimeric NKp30 mouse T cells specificallyrespond to tumor cells expressing NKp30 ligands. These results indicatethat Human NKp30 Receptors are functional in mouse T cells.

Effector T cells derived from B6 (open), perforin-deficient (Pfp−/−,filled) mice that were modified with NKp30 receptors were co-culturedwith RMA or RMA/B7-H6 cells, respectively, at a ratio of 1:1 5-hr LDHrelease assays. The data are presented as mean±SD of triplicates and arerepresentative results from two independent experiments. The T cellslysed a significantly higher percentage of NKp30 ligand-positive cells(RMA/B7-H6, FIG. 10B) than ligand-negative cells (cell line RMA, FIG.10A). Specific lysis was substantially decreased with the Pfp−/−cells.These results demonstrated that specific lysis of RMA/B7-H6 byNKp30-modified murine T cells required perforin.

Example 5: Chimeric Human NKp30 Receptors Leads to Better Anti-Tumor InVivo Efficacy and Enhanced Mouse Survival in a Murine B7-H6+ LymphomaModel

In this example, mouse T cells transduced with chimeric NKp30 were shownto enhance survival of mice injected with lymphoma cells, specificallyRMA/B7-H6 that express the NKp30 ligand B7-H6 (schematically illustratedin FIG. 11A). Integration of CD28 TM and signaling domains into chimericNKp30 receptor was demonstrated to lead to better anti-tumor in vivoefficacy. RMA/B7-H6 cells (10C) were administered i.v. on day 0. On day5, 7 and 9, tumor-bearing mice were injected with T cells (5×10⁶) thatwere modified to express wtNKp30 (♦), NKp30-CD3ζ (▴), NKp30-CD8-CD3ζ (●)and NKp30-CD28-CD3ζ (□), respectively (FIG. 11B). Injection with asaline solution (HBSS) was used as negative control. Data are presentedin Kaplan-Meier survival curves.

Previous results had shown that i.v. injection of RMA cells leads tosystemic lymphoma in B6 mice (Zhang et al., Cancer Res. 67:11029-11036). The present results demonstrate that although B7-H6 is ahuman molecule, its expression did not significantly alter the growth ofRMA cells. Intravenous administration of 10⁵ RMA/B7-H6 cells led to thedevelopment of systemic lymphoma, with a median survival of 18 d, whichis the typical growth for a similar dose of RMA tumor cells in B6 mice(Zhang et al., Cancer Res. 67: 11029-11036).

Survival curves were similar for mice injected with saline or T cellstransduced with wtNKp30 (with no mice surviving past about 20-22 days)indicating that wtNKp30 T cells had little or no beneficial effect.Additionally, although wtNKp30, NKp30-3ζ, and NKp30-CD8(TM)-3ζ allowed Tcells to respond to RMA/B7-H6 cells in vitro, the murine T cellsmodified with these receptors showed little effect on the survival oftumor-bearing mice in this aggressive lymphoma model.

Slight improvements in survival were obtained with NKp30-CD3ζ T cells,particularly for the final 25% of surviving mice, with some micesurviving for up to about 30 days. Further improvements in survival wereobtained with NKp30-CD8-CD3ζ T cells, with some mice surviving for up toabout 42 days. However, the greatest improvements in survival wereobserved with NKp30-CD28-CD3ζ T cells. Treatment with NKp30-CD28-3ζ+ Tcells significantly improved median survival from 18 to 30 d, withapproximately 20% of the population (4 out of 23 mice) surviving to theend of the experiment (60 days). Several of these mice become long-termsurvivors, indicating that treatment with this chimeric NKp30 T cellresulted in tumor eradication.

Example 6: Chimeric NKp30-CD28-CD3ζ T Cells Confer Long-Term TumorResistance

In the preceding example, several of the mice treated withNKp30-CD28-CD3ζ T cells became long-term survivors subsequent toinjection with lymphoma cells. The lymphoma cells were RMA cellstransformed with a B7-H6 construct (RMA/B7-H6).

To determine whether this chimeric NKp30 T cell resulted inNKp30-independent tumor immunity, long-term survivors were re-challengedwith a similar, but ligand-deficient, lymphoma, specifically RMA cellsthat had not been transformed with the B7-H6 construct. These survivorswere indeed resistant to the tumor re-challenge. Overall survival wasdetermined, and none of the re-challenged mice had tumor growth by theend of the study period, whereas naïve mice did (FIG. 11C). Theseresults show that this chimeric T cell treatment can lead to tumoreradication and suggest induction of long-term tumor immunity can beacquired.

Because ligand-negative tumor cells could selectively grow out after CART cell therapy, it would be beneficial if treatment with NKp30-CD28-3δ+T cells induced host immunity against other tumor antigens. Theseresults demonstrate that adoptive transfer of NKp30-CD28-3ζ+ T cells mayallow hosts to generate immunological memory against RMA tumor antigens.In addition, we observed that NKp30-CD28-3ζ+ T cells persisted longerthan did either NKp30-3ζ+ or NKp30-CD8(TM)-3ζ+ T cells (data not shown),which correlated with their enhanced antitumor efficacy.

Example 7: Additional Exemplary Embodiments of Chimeric NKp30 Receptors

Additional chimeric NKp30 receptors were produced as illustrated inFIGS. 18 and 20-22. These sequences encoding these receptors wereintroduced into human and/or mouse T cells, and their surface expressionwas detected (FIGS. 3 and 9). T cells transformed with these constructswere also demonstrated to produce IFN-γ production and cause specificlysis of NKp30 ligand-positive tumor cells (FIGS. 6 and 7). These cellsare tested in vitro and/or in vivo for cancer cell killing activity asdescribed above in Examples 5 and 6.

Example 8: Human DCs Express NKp30 Ligands and can Stimulate ChimericNKp30-Expressing T Cells to Produce IFN-7

This example shows that PBMC-derived dendritic cells express NKp30ligands, including immature dendritic cells, and they can stimulateNKp30 CAR-bearing T cells to produce IFN-γ, but to a lesser extent thanNKp30 ligand-positive tumor cells.

It is known that NKp30 plays an important role in human NK-DCinteractions (Moretta t al., Immunol. Lett. 100: 7-13; Vitale et al.,Blood 106: 566-571). At low NK/DC ratios, DCs promote IFN-γ productionand cytotoxicity by NK cells in an NKp30-dependent manner (Vitale etal., Blood 106: 566-571; Ferlazzo et al., J. Exp. Med. 195: 343-351),which suggests that DCs express NKp30 ligands. With the use ofNKp30-mIg2a, we confirmed that both iDCs and mDCs can bind to solubleNKp30, which is consistent with DCs expressing ligands for NKp30 (FIG.12A). The level of cell surface staining on iDCs was higher than onmDCs. However, there was no significant expression of B7-H6 on DCs asdetermined with mAb 47.39, a specific anti-B7-H6 mAb. To determinewhether the NKp30 CAR-modified T cells can respond to DCs, T cells werecocultured with either iDCs or mDCs at a 5:1 ratio for 24 h. IFN-7production (200-800 pg/ml) was observed by NKp30-28-C-modified T cellsafter coculture with DCs (FIG. 12B). Compared with mDCs, iDCs inducedhigher amounts of IFN-γ, which reflected their greater binding tosoluble NKp30.

Example 9: Integration of a CD28 Signal into the NKp30 Chimeric AntigenReceptor Promotes In Vitro T Cell Proliferation

It is known that CD28 signals enhance T cell survival and proliferation.To Investigate whether integration of CD28 signaling in the chNKp30receptor resulted in similar outcomes upon engagement of the NKp30receptor, CFSE-labeled T cells were cocultured with HeLa cells (NKp30ligand positive) in the presence of a small amount of IL-2 (25 U/ml) for3 d. As shown in FIG. 13A, engagement of NKp30-CD28-3ζ-bearing T cellsled to more T cell proliferation than did engagement of NKp30-3ζ-bearingT cells. Two important mechanisms through which CD28 signaling promotesT cell survival are through upregulation of IL-2 and an antiapoptoticprotein, Bcl-xL. To determine whether NKp30-CD28-3ζ+ T cells upregulatedIL-2 and Bcl-xL in response to cross-linking of the chimeric receptor, Tcells were cultured in anti-NKp30 mAb-coated wells for 24 h. IL-2 mRNAand Bcl-xL protein were determined using real-time PCR and intracellularstaining, respectively. The results show that NKp30-CD28-3ζ+ T cellsincreased IL-2 expression by 25-fold after receptor crosslinkingcompared with a 10-fold induction in NKp30-3ζ+ T cells (FIG. 13B). Nosignificant upregulation of IL-2 was observed in wtNKp30-modified Tcells cultured under these conditions. Similarly, we observed greaterexpression of Bcl-xL in NKp30-CD28-3ζ+ T cells compared with NKp30-3ζ+ Tcells (FIG. 13C). These data suggest that NKp30-CD28-3ζ+ T cells canreceive a costimulatory signal through CD28 that leads to increased IL-2and Bcl-xL expression.

Example 10

Surface expression of chimeric NKp30 receptors on human T cells wasanalyzed by flow cytometry using anti-NKp30 and anti-CD4 mAbs. As shownin FIG. 3K, retroviral transduction of human T cells with wtNKp30 genedid not lead to significant surface expression. Insufficient expressionof wtNKp30 on the cell surface may be due to an absence of FcgRexpression on T cells, which is associated with CD3γ and NKp30 on humanNK cells. Replacement of the NKp30 TM domain with the CD3γ TM domainimproved CAR expression. Our results showed that the CD8a TM domainefficiently allows surface expression of chimeric NKp30 receptor on Tcell. The human CD28 TM domain also resulted in higher surfaceexpression of chimeric NKp30, with or without the CD28 CYP domain.

Chimeric NKp30-Expressing T Cells Produce IFN-γ Upon Coculture withNKp30 Ligand-Positive Tumor Cells

A panel of human tumor cell lines was screened for NKp30 ligandexpression using a soluble human NKp30-mIgG2a fusion protein and ananti-B7-H6 mAb (called 47.39). As shown in FIG. 4B, K562, A375, HeLa,and T47D cells expressed high amounts of NKp30 ligands, whereas U937,RPMP8226, DU145, and Panc-1 cells expressed low amounts of NKp30ligands. Some tumor cells (IM9, U266, and MCF-7) did not express NKp30ligands on the cell surface. All tested human tumor cells havedetectable levels of BAT3 mRNA, as detected by RT-PCR (FIG. 4B). Incontrast, B7-H6 mRNA levels were correlated to surface expression ofNKp30 ligands, suggesting that B7-H6 is the major surface ligand ofNKp30 in tumors. The result was also consistent with the fact that BAT3is a nuclear protein, which is usually not expressed on the cellsurface. In addition, RT-PCR results showed that PBMCs lacked the mRNAsof B7-H6 and Bat3 (FIG. 4B).

To determine whether chimeric NKp30-transduced human T cells were ableto recognize NKp30 ligand-positive tumor cells, the chimeric NKp30CAR-bearing T cells were cultured with different tumor cells, and IFN-γresponses measured by ELISA. As shown in FIGS. 6D-E, NKp30-3ζ+,NKp30-CD8(TM)-3ζ+, NKp30-CD28(TM)-3ζ+, or NKp30-CD28-3ζ+ T cellsproduced significant amounts of IFN-γ after coculture with NKp30ligand-positive cells but not when cultured with ligand-negative cells,indicating that these NKp30 CAR-modified T cells could functionallyrecognize NKp30 ligand-bearing tumor cells. In contrast,wtNKp30-modified T cells did not show any significant response to thestimulation by NKp30 ligand-positive cells. NKp30-CD28-3ζ-expressing Tcells produced significantly more IFN-γ than did T cells expressingNKp30-3ζ+, NKp30-CD8(TM)-3ζ+, or NKp30-28 (TM)-3ζ+ in the presence ofNKp30 ligand-positive tumor cells, especially when the ligand expressionwas low. The reason was likely due to higher surface expression of thisCAR and/or the presence of the CD28 costimulatory signaling domain inNKp30-CD28-3.

Chimeric NKp30-Bearing Human T Cells Kill NKp30 Ligand-Positive TumorCells

The cytotoxic activity of chimeric NKp30-modified human T cells againstvarious tumor cell lines was determined. As shown in FIG. 7C, NKp30CAR-bearing T cells were able to lyse NKp30 ligand-positive target cells(RMA/B7-H6, T47D, Panc-1, A375, K562, and RPMI8226) but not theligand-negative cell lines RMA and MCF-7 in vitro. Similar to cytokineproduction, no significant killing was observed when wtNKp30-modified Tcells were used. NKp30-CD28-3ζ receptor bestowed T cells withsignificantly higher lytic activity than did NKp30-3ζ, NKp30-CD8(TM)-3ζ,or NKp30-CD28(TM)-3ζ. Because RMA/B7-H6 tumor cells lack expression ofhuman MHC class I and II molecules, these data indicate that thechimeric NKp30 receptor-modified T cell-mediated killing of these tumorcells was ligand dependent and MHC independent. To confirm that chimericNKp30-mediated killing is dependent on the interactions between NKp30and its ligands, soluble NKp30 (NKp30-mIgG2a) was incubated with K562target cells prior to the coculture with T cells. As shown in FIG. 7C,NKp30-mIgG2a significantly reduced NKp30-28-3ζ-bearing T cell-mediatedcytotoxicity. These results demonstrated that chimeric NKp30-bearing Tcells killed ligand-positive tumor cells, and the interaction betweenchimeric NKp30 receptors and NKp30 ligands was essential for chimericNKp30-mediated T cell function.

Adoptive Transfer of NKp30-CD28z+ T Cells Significantly Improves theSurvival of RMA/B7-H6 Tumor-Bearing Mice and Induces the Generation ofImmunological Memory

Because NKp30 is a pseudogene in inbred mice, we determined whetherhuman NKp30 CARs could be expressed on mouse T cells, which allows thetesting of in vivo efficacy of chimeric NKp30-modified T cells againstNKp30 ligand-positive tumor cells in immunocompetent mouse tumor models.Similar to the expression profile observed on human T cells, NKp30 CARswere expressed on mouse T cells (FIG. 9E-J). All chNKp30-modified murineT cells responded to coculture with RMA/B7-H6 cells, but not with RMAcells, by producing IFN-γ; NKp30-CD28-3-bearing T cells produced moreIFN-7 than did NKp30-3-expressing T cells (FIG. 10C). wtNKp30-modifiedmouse T cells also produced low levels of IFN-γ in response to B7-H6(FIG. 1C). In addition, chNKp30-modified mouse T cells were highlycytotoxic. Even at an E:T ratio of 1:1, T cells expressing eitherNKp30-CD28-3ζ or NKp30-3ζ receptor killed RMA/B7-H6 cells at anefficiency of 80% (FIG. 10A-B). No significant killing of NKp30ligand-negative RMA cells was observed. Perforin had a role in thekilling process, because NKp30 CAR-modified T cells deficient inperforin showed a significantly reduced (p<0.05) ability to killRMA/B7-H6 tumor cells.

Methods

Except where indicated otherwise, the experimental results weregenerally obtained using the methods that follow.

Mice

C57BU6 (B6; wild-type [wt]) mice were purchased from the National CancerInstitute (Frederick, Md.). Perforin-deficient mice C57BJ6-Prf1tm1Sdz/J(Pfp2/2) were obtained from The Jackson Laboratory (Bar Harbor, Me.).All experiments were conducted according to protocols approved byDartmouth College's Institutional Animal Care and Use Committee.

Cell Lines and Cell Culture

Bosc23, GP+E86, PT67, K652, U937, HeLa, U266, and Jurkat cell lines wereobtained from the American Type Culture Collection (Rockville, Md.).Breast cancer cell lines MCF-7 and T47D were provided by Dr. JamesDirenzo (The Geisel School of Medicine at Dartmouth). Pancreatic cancercell line Panc-1 was provided by Dr. Murray Korc (School of Medicine,Indiana University, Indianapolis, Ind.). Prostate cancer cell line DU145and melanoma cell line A375 were provided by Dr. Marc Ernstoff (TheGeisel School of Medicine at Dartmouth). An RMA subline RMA/B7-H6 thatexpresses an NKp30 ligand, B7-H6, was generated by retroviraltransduction using dualtropic retroviral vectors containing the B7-H6gene, according to our previous protocol (Zhang et al., Blood 106:1544-1551). Packaging cells Bosc23, GP+E86, and PT67 were grown in DMEMwith a high glucose concentration (4.5 g/l), supplemented with 10%heat-inactivated FBS (Atlanta Biologicals, Lawrenceville, Ga.), 100 U/mlpenicillin, 100 μg/ml streptomycin, 1 mM pyruvate, 10 mM HEPES, 0.1 mMnonessential amino acids, and 50 μM 2-ME. All other cell lines werecultured in RPMI 1640 plus the same supplements as in DMEM. Humandendritic cells (DCs) were generated from blood mononuclear cells thatwere obtained from cell cones from the Dartmouth-Hitchcock MedicalCenter Blood Donor Center from leukapheresis cell donations. CD14+ cellswere selected using magnetic beads (Miltenyi Biotec) and were culturedin six-well plates (5×10⁵/ml) in 2 ml complete RPMI 1640 media withrecombinant human IL-4 (100 ng/ml; PeproTech, Rocky Hill, N.J.) andrecombinant human GM-CSF (100 ng/ml; PeproTech). On days 4 and 6, 2 mlfresh media with IL-4 and GM-CSF was added to the cultures. On day 8,the nonadherent cells were collected and used as iDCs. To generate mDCs,the media were replaced with fresh media containing LPS (1 μg/ml; Sigma,St. Louis, Mo.) and CD40L (200 ng/ml; PeproTech) on day 6 for 2 d.

Construction of Chimeric NKp30 Receptors

The full-length human NKp30, CD28, and CD8a cDNAs were purchased fromOpen Biosystems (Huntsville, Ala.). Human CD3γ-chain-signaling domainand full-length B7-H6 cDNAs were cloned by RT-PCR using RNAs from Jurkatcells as templates. The chimeric NKp30 constructs used in this study areillustrated in FIGS. 2 and 12. wt NKp30 contains the full-length humanNKp30. Chimeric receptor NKp30-3ζ comprises the extracellular domain (aa1-139) of human NKp30 fused to the TM domain (aa 31-51) and thesignaling domains of human CD3γ-chain (aa 52-164). The NKp30-CD8-3ζreceptor was constructed by joining the extracellular domain of NKp30 tothe TM domain of human CD8a (aa 183-203) and the signaling domain ofCD3γ-chain. The NKp30-CD28-34 receptor contains the TM and signalingdomains of CD28 (aa 153-220) and the signaling domain of the CD3γ-chain.As a control receptor, NKp30-28(TM)-3ζ is similar to NKp30-CD28-3ζ,except that the CD28-signaling domain was removed. All PCR reactionswere performed using a high-fidelity DNA polymerase Phusion (New EnglandBioLabs, Ipswich, Mass.). All oligonucleotides were synthesized bySigma-Genosys (The Woodlands, Tex.). All genes were cloned into aretroviral vector pFB-neo (Stratagene, Palo Alto, Calif.).

Retroviral Transduction

Production of retroviral vectors and retroviral transduction wereperformed according to modified protocols, as described previously(Zhang et al., Blood 106: 1544-1551; Zhang et al., Cancer Res. 66:5927-5933). In brief, transduction of murine primary T cells wasconducted using ecotropic viruses collected from vector-transfectedGP+E86 cells, whereas dualtropic retroviral viruses generated fromvector-transfected PT67 cells were used to infect human primary T cells.Primary T cells from spleens of B6 mice were infected 18-24 h after ConA (1 μg/ml; Sigma) stimulation. Two days postinfection, transducedprimary T cells (0.5-1×10⁶/ml) were selected in RPMI media containingG418 (1 mg/ml) plus 25 U/ml recombinant human L-2 for three additionaldays. Viable cells were isolated using Histopaque-1083 (Sigma), washedextensively, and expanded for 2 d without G418 before functionalanalyses or i.v. injection. Primary human cells were stimulated withanti-CD3 mAb OKT3 (40 ng/ml; eBioscience, San Diego, Calif.) for 3 dbefore retroviral transduction. 0418 selection of retrovirallytransduced human T cells followed the same procedures for selectingchimeric antigen receptor-transduced murine T cells.

Production of Soluble Human NKp30-mIgG2a Fusion Protein

To make a soluble human NKp30-mIgG2a fusion protein, the extracellularportion of human NKp30 (aa 1-262) was fused to the mouse IgG2ahinge-CH2-CH3 portion. NKp30-mIgG2a gene was cloned into pFB-neo.NKp30-mIgG2a fusion protein was expressed in retroviral vector stablytransduced B16F10 cells. The production and purification of NKp30-mIgG2aprotein were performed according to previous protocols (Zhang et al.,Blood 106: 1544-1551; Zhang et al., Cancer Res. 66: 5927-5933).

Generation of Anti-B7-H6 mAbs

Eight- to twelve-week-old B6 mice were immunized (i.p.) with mitomycinC-treated RMA/B7-H6 cells (5×10⁶). Two weeks after the initialimmunization, mice were boosted with 50 μg recombinant B7-H6(extracellular domain) prepared from Escherichia coli and IFA (Sigma),three times at weekly intervals. Three days after the last boostingimmunization, mice splenocytes were fused to NS1 cells (provided by Dr.William Wade, The Geisel School of Medicine, Dartmouth College) usingstandard techniques, and hybridoma supernatants were screened forreactivity to both B7-H6-negative and -positive cell lines by flowcytometry. Two clones (47.39 and 127.4; both of the Ig2a subclass) wereisolated.

Flow Cytometry

For flow cytometry analysis of NKp30 ligand expression, cells werestained with either NKp30-mIgG2a or anti-B7-H6 mAb, followed by DyLight649-conjugated goat anti-mouse IgG (BioLegend, San Diego, Calif.). Cellsurface phenotyping of transduced primary T cells was determined bystaining with FITC-conjugated anti-CD4 (clone OKT4; BioLegend),PEconjugated anti-NKp30 (clone P30-15; BioLegend) mAbs. The followingmAbs were used to analyze the cell surface phenotype of DCs:allophycocyanin-conjugated anti-CD86 (BioLegend), PE-conjugatedanti-CD11c (BioLegend), FITC-conjugated anti-CD83 (BioLegend), andFITCconjugated anti-HLA-DR (eBioscience). Intracellular staining ofBcl-xL in T cells was performed using anti-Bcl-xL-FTC (Southern Biotech,Birmingham, Ala.), based on the protocol described previously (Erikssonet al., J. Leukoc. Biol. 76: 667-675). All samples were preincubatedwith either FcR block Ab (anti-mouse CD16/CD32, 2.4G2; Bio X Cell,Lebanon, N.H.) for mouse cells staining or human g globulins (Cohn'sfraction, G4386; Sigma) for human cell staining. Cell fluorescence wasmonitored using an Accuri C6 cytometer. Flow cytometry analysis wasperformed using either Accuri or FlowJo software.

RT-PCR and Quantitative PCR

Extraction of total RNA and preparation of cDNAs from human tumor celllines and PBMCs were performed as described (Zhang et al., Cancer Res.66: 5927-5933). The resulting cDNA, corresponding to 50 ng total RNA,was subjected to PCR amplification in a total volume of 20 ml, including0.5 mmol/I each primer, 0.2 mmol/I each deoxynucleotide triphosphate,and 1 U Taq DNA polymerase (New England BioLabs). The primers used foramplification are shown in Supplemental Table I. The PCR conditions wereas follows: 95° C. for 5 min, followed by 30 cycles of 95° C. for 30 s(denaturation), 60° C. for 30 s (annealing), and 72° C. for 30 s(extension), with a 3-min incubation at 72° C. at the end. The PCRproducts were run on agarose gels and visualized by staining with SYBRSafe (Invitrogen). For quantitative real-time PCR of human IL-2 mRNA,triplicates of cDNA samples from T cells were mixed with SYBR GreenMaster Mix (Applied Biosystems) and IL-2-specific primers (SupplementalTable I) in a total volume of 25 ml. The reactions were run on a Bio-RadiCycler. The relative gene expression was calculated as described(Erikkson et al., J. Immunol. 176: 6219-6224). GAPDH gene was used as aninternal control. The mean value of relative IL-2 expression in controlmAb-treated T cell samples was set as 1.

Cytotoxicity Assay

Cytotoxicity of T cells against target cells was determined by anLDHrelease assay using the CytoTox 96 Non-Radioactive Cytotoxicity Assaykit (Promega, Madison, Wis.). Specific lysis was determined using thefollowing equation: percentage of specific lysis=[(experimental−effectorspontaneous−target spontaneous)/(target maximum−targetspontaneous)]×100. In the cytotoxicity-blocking experiments, K562 targetcells were preincubated with a soluble NKp30 receptor, NKp30-mIgG2a (10μg/ml), for 30 min before coculture with T cells in an LDH-releaseassay.

Cytokine Production by T Cells

To determine whether chimeric antigen receptor T cells responded totumor cells with production of IFN-γ, T cells (10⁵) were cocultured withsuspension tumor cells at an E:T ratio of 1:1 or with adherent tumorcells at an E:T ratio of 1:0.25 (10⁵:2.5×10⁴) in 96-well V-bottom orflat-bottom plates, respectively, for 24 h. Cell-free supernatants wereassayed for IFN-γ by ELISA using DuoSet ELISA kits (R&D Systems).

Treatment of Lymphoma-Bearing Mice with Chimeric NKp30-Modified T Cells

As a systemic mouse lymphoma model, B6 mice were injected with 10⁵RMA/B7-H6 cells in 400 ml HBSS via tail veins. For treatment with Tcells, mice were administered 5×10⁶ wt NKp30 (wtNKp30) or chimericNKp30-modified T cells i.v. starting on day 5 posttumor inoculations. Tcell transfer was repeated on days 7 and 9 using T cells from the same Tcell preparation that were expanded for additional days in vitro. Micewere monitored closely and sacrificed when they became moribund.

Tumor Rechallenge

Mice that survived for 60 d after initial tumor inoculation withoutsigns of disease were regarded as tumor free and were inoculated s.c.with 10⁴ wt RMA tumor cells on the shaved right flank. Naive B6 micewere used as controls. Tumor size was monitored every 2 d, and mice weresacrificed when tumor burden became excessive.

Statistical Analysis

Differences between groups were analyzed using a Student t test orANOVA; p values <0.05 were considered significant. Kaplan-Meier survivalcurves were plotted and analyzed using Prism software (GraphPadSoftware, San Diego, Calif.).

1. A nucleic acid construct for expressing a chimeric receptorcomprising: a first nucleic acid sequence comprising a promoter operablylinked to a second nucleic acid sequence, wherein said second nucleicacid sequence encodes a chimeric NKp30 receptor comprising: an NKp30extracellular domain, a transmembrane domain, and at least one signalingdomain. 2-394. (canceled)